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BioProbes 73

thermofisher.com/bioprobes • May 2016

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Published by Thermo Fisher Scientific Inc. © 2016

BioProbes Journal, available in print and online at thermofisher.com/bioprobes, is dedicated to providing researchers with the very latest information about cell biology products and their applications. For a complete list of our products, along with extensive descriptions and literature references, please see our website.

EditorsMichelle Spence

Grace Richter

DesignerLynn Soderberg

ContributorsLaura Allred

Brian Almond

Chandan Bhambhani

Jolene Bradford

Beth Browne

Suzanne Buck

Ankita Chiraniya

Scott Clarke

William Dietrich

Carmen Finnessy

Jane Helmer

Athena Jagdish

Kamran Jamil

Mike Janes

Navin Jose

Greg Kaduchak

Jason Kilgore

Jason Kim

Tom Landon

Chris Langsdorf

Victoria Love

Pradeep Narayan

Monica O’Hara

Thao Sebata

Deepa Shankar

Laura Shapiro

Matt Slater

Brian Steer

Mike Thacker

Niveditha Vathsangam

Production ManagerBeth Browne

ONLINE AND ON THE MOVE

2 | Cell Analysis Support Center, updated mobile apps, virtual training labs, and more

JUST RELEASED

4 | Our newest cellular analysis products and technologies

FEATURED RESEARCH TOOLS

6 | Optimize immunodetection with recombinant secondary antibodiesSuperclonal secondary antibodies for sensitive and reproducible immunoassays

PROTEIN FUNCTION AND ANALYSES

10 | Cells and soluble mediators of the tumor microenvironmentAntibody-based tools for identifying cell types and proteins of interest

12 | Monitor changes in gene expression using luminescenceThe HTS-compatible TurboLuc Luciferase One-Step Glow Assay

14 | Cast your protein gels with easeIntroducing the SureCast Gel Handcast System for protein separation

15 | Load more sample with these precast protein gelsNovex WedgeWell Tris-glycine precast protein gels

CELL VIABILITY AND PROLIFERATION

17 | Multiplex assays for robust cell health analysesCyQUANT Direct and PrestoBlue viability assays work together

20 | Proliferating and apoptotic cells revealedClick-iT EdU and Click-iT TUNEL colorimetric assays for immunohistochemistry

24 | Accurately measure cell concentrations by flow cytometryHigh-speed cell counting with the Attune NxT Flow Cytometer

26 | Protection from photobleaching for live-cell imagingProLong Live Antifade Reagent is here

27 | Tracking cell division with flow cytometryA guide to studying cell proliferation by generational tracing

JOURNAL CLUB

30 | Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

31 | Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments

IN MEMORIAM

32 | A tribute to our colleague Mike Davidson

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2 | thermofisher.com/bioprobes © 2016 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.

ONLINE AND ON THE MOVE BIOPrOBEs 73

We recently launched our 14th application-specific support center—this one focused on cell

analysis. At the Cell Analysis Support Center, you’ll find 12 hubs covering the following topics:

■ Labeling chemistry

■ Microspheres

■ Cell imaging

■ Cell structure and analysis—

ICC, IHC, and IF

■ Flow cytometry

■ Microbiological analysis

■ Quantum dots

■ Cell counting

■ Cell viability, proliferation, cryopreservation,

and apoptosis

■ Cell tracing and tracking

■ Neuroscience

■ Dynabeads™ cell isolation and expansion

Additional support centers—including those for protein electrophoresis and western blotting,

protein expression, and genome editing—can be found on the Services and Support tab at

thermofisher.com or at thermofisher.com/supportcenters. These Support Centers have

been created by our technical applications scientists to answer the most commonly asked

questions for every step of your experimental workflow, from setting up and running an exper-

iment to analyzing data and troubleshooting. Visit the Cell Analysis Support Center today at

thermofisher.com/cellanalysissupport.

The new Applied Biosystems™ LabCoat Live™ Training Series is specifically designed to fit

into your everyday lab life, whether you are a lab manager who wants help training your staff

or a bench scientist conducting research in the lab. LabCoat Live courses simplify complex

concepts through a combination of live, online classroom lectures and self-paced lab time

over the span of one week. Now you don’t need to take time away from your projects to train

others or travel to a training center. LabCoat Live training makes learning new techniques both

affordable and convenient with:

■ Two interactive online lectures, providing a thorough understanding of technique, experiment

setup, and data analysis so that you can review your results in real time from anywhere

with an internet connection

■ An online message board, with step-by-step experiment setup guides to get your questions

answered quickly

■ Reagents and a protocol provided to conduct the experiment on your own instrument at

a time that works for you

Specifically, the “Introduction to qPCR” module focuses on qPCR fundamentals, applications,

and data analysis, including how to set up and perform an absolute quantitation experiment

and then analyze standard curve data using Applied Biosystems™ software. Register for an

upcoming class today at thermofisher.com/labcoatlive (not available in all regions).

LabCoat LiveA uniquely interactive virtual training program

LabCoat Live qPCR Training Series: Interactive online lectures with a self-paced lab

Introducing our Cell Analysis Support Center

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http://thermofisher.com/supportcenters

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BIOPrOBEs 73 ONLINE AND ON THE MOVE

Our new handbook series covers topics in these research areas: cancer signaling, tumor

inflammation, stem cell research, and neurodegenerative disease research—with particular

emphasis on evaluating protein biomarkers. For each topic, we provide:

■ A brief overview of the current state of research

■ Antibody-based resources, including multiplex characterization of critical proteins

■ Access to detailed protocols for a wide range of experimental techniques, including west-

ern blotting, immunofluorescence analysis, flow cytometry, enzyme-linked immunosorbent

assays (ELISAs), and more

Download one or all of these technical handbooks by simply completing the online form at

thermofisher.com/abtoolshandbooks.

Updated mobile apps move to Thermo Fisher Scientific

All of the former Life Technologies mobile apps (both on Google Play™ and at iTunes™) now

live under Thermo Fisher Scientific, including all of the Molecular Probes™ educational apps,

as well as those associated with Invitrogen™ and Applied Biosystems™ brands. Check out the

new look and feel of these updated mobile apps for cell analysis:

■ 3D Cell

■ Cell Imaging Reagent Guide & Protocols

■ Flow Cytometry Reagent Guide & Protocols

■ Fluorescence SpectraViewer

Additionally, all of the iTunes apps have been upgraded to iOS 9. Download these apps

today at the App Store™ (for iPhone™ and iPad™ devices) or at Google Play™ (for Android™

phones and tablets), and see our complete selection of mobile and desktop apps at

thermofisher.com/mobileapps.

Virtual training labs: Cell culture basics and more

Cell culture is critical for cell biology research, and your experiments rely on your ability to

produce and maintain consistently healthy cells. The Cell Culture Basics virtual training lab is

an introduction to cell culture that covers many topics, including:

■ Basic principles of cell culture

■ Preparing a new cell culture

■ Performing a cell passage

■ Cryopreservation

This free, 3D interactive learning laboratory offers cell culture training modules, best practices

for working with your cells, and quizzes to test your understanding. Make sure you are using

state-of-the-art products and procedures for your cell culture by registering for our Cell Culture

Basics virtual training lab at thermofisher.com/gibcoeducation. There you will find several

other virtual training labs, including those on pluripotent stem cell culture, transfection, and

protein expression.

Free handbook series: Antibody-based tools for biomedical research

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JUsT rELEAsED BIOPrOBEs 73

Click-iT Plus EdU Assay Kits: Now with Alexa Fluor 350 and Alexa Fluor 594 dyes

Human Adipokine 14-Plex Magnetic Panel for the Luminex platform

To expand the multiplexability of the Invitrogen™ Click-iT™ Plus EdU

assay for flow cytometry, we have recently introduced kits with either

Alexa Fluor™ 350 or Alexa Fluor™ 594 dye, which allow detection of

the EdU signal using a UV or 532/561 nm laser, respectively. These

two kits join our family of Click-iT Plus EdU Flow Cytometry Assay

Kits (see product list), providing maximum flexibility when designing

an EdU-based cell proliferation experiment.

The Click-iT EdU cell proliferation assay utilizes click chemistry

to provide a superior alternative to traditional BrdU methods for

labeling newly synthesized DNA. When the modified nucleoside EdU

(5-ethynyl-2´-deoxyuridine) is incorporated during DNA synthesis, it

can be detected by a quick click reaction, with minimal disruption

to the cell. Unlike antibody-based BrdU assays, Click-iT Plus EdU

assays employ a small fluorescent picolyl azide for detection of the

incorporated EdU and thus do not require harsh DNA denaturation

treatments. Furthermore, these fluorescence-based Click-iT Plus EdU

assays can be multiplexed with fluorescent proteins such as R-PE,

R-PE tandems, and GFP. The Alexa Fluor 350 kit can be multiplexed

with other violet-excitable dyes such as Pacific Orange™ conjugates,

which emit at ~550 nm. Learn more about the Click-iT EdU cell pro-

liferation assays for flow cytometry at thermofisher.com/clickitflow.

Detection of cell proliferation with Click-iT Plus EdU Flow Cytometry Assay

Kits. Jurkat (human T-cell leukemia) cells were treated with 10 μM EdU and detected

according to the recommended Click-iT™ Plus EdU staining protocol. The figures each

show a clear separation of nonproliferating cells and proliferating cells, which have

incorporated EdU and been labeled with either (A) Alexa Fluor™ 350 picolyl azide or

(B) Alexa Fluor™ 594 picolyl azide.

Adipokines—diverse peptides secreted by adipose (fat) tissue—are

a group of cytokines, hormones, and other signaling proteins that

play an important role in many health problems, including obesity,

diabetes, metabolic disorders, and immune-related diseases.

The Invitrogen™ Human Adipokine 14-Plex Magnetic Panel for the

Luminex™ platform enables researchers to quickly measure multiple

proteins in one well in order to achieve a more holistic understanding

of cell signaling interactions. Requiring only 25 µL of serum per well,

this Luminex assay panel interrogates 14 targets in a single day. Not

only is multiplexing efficient, it also reduces the variability of running

assays on different days and samples. This panel is suitable for use

with the Luminex™ 200™, FLEXMAP 3D™, and MAGPIX™ systems.

Find more Luminex™ assay panels at thermofisher.com/luminex.

Measurement of serum levels of 14 protein targets in one assay run. Sera from

normal and diseased patients were tested with the Human Adipokine 14-Plex Magnetic

Panel using the Luminex™ 200™ System.

Click-iT Plus EdUAlexa Fluor 594 �uorescence

Num

ber

of c

ells

102 103 104 105

Green or yellow laser

Click-iT Plus EdUAlexa Fluor 350 �uorescence

Num

ber

of c

ells

0–103 103 104 105

UV laserBA

Product Quantity Cat. No.

Click-iT™ Plus EdU Alexa Fluor™ 350 Flow Cytometry Assay Kit 50 assays C10645

Click-iT™ Plus EdU Alexa Fluor™ 594 Flow Cytometry Assay Kit 50 assays C10646

Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit 50 assays 100 assays

C10632 C10633

Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit 50 assays 100 assays

C10634 C10635

Click-iT™ Plus EdU Pacific Blue™ Flow Cytometry Assay Kit 50 assays C10636

Product Targets Quantity Cat. No.

Human Adipokine 14-Plex Magnetic Panel BAFF, IL-1β, IL-6, IL-8, IL-10, HGF, insulin, leptin, MCP-1, lipocalin-2, resistin, PAI-1, SAA, TNF-α 100 tests LHC0017M

Con

cent

ratio

n (p

g/m

L)

BA

FF

IL-1β

IL-6

IL-8

IL-1

0

MC

P-1

Lep

tin

HG

F

Insu

lin

Lip

ocal

in-2

PAI-

1

SA

A

TNF-α

Res

istin

101

102

103

104

105

106

NormalObesityDiabetesSLEAsthma

http://thermofisher.com/clickitflow

http://thermofisher.com/luminex


BIOPrOBEs 73 JUST RELEASED

We offer a wide range of epitope-tag and loading-control antibodies conjugated with Invitrogen™

Alexa Fluor™ dyes, including green-fluorescent Alexa Fluor 488, orange-fluorescent Alexa Fluor

555, and far red–fluorescent Alexa Fluor 647 dyes. The epitope-tag antibodies are highly specific

monoclonal and polyclonal antibodies that recognize commonly used epitopes, including FLAG,

GST, HA, His, Myc, and V5. The loading-control antibodies recognize the most common loading

and expression control proteins, including GAPDH, β-actin, and β-tubulin. They are used to

normalize signals on western blots in order to distinguish expression level differences from loading

variability in the samples, and also as complementary stains in immunofluorescence studies.

In addition to the Alexa Fluor conjugates, the epitope-tag and loading-control antibodies

are available unconjugated or conjugated to biotin, horseradish peroxidase (HRP), or Thermo

Scientific™ DyLight™ dyes. See the complete selection of epitope-tag and loading-control

antibodies at thermofisher.com/tagabs and thermofisher.com/loadingctrlabs.

The Invitrogen™ ArC™ Amine-Reactive Compensation Bead Kit, now available in two sizes, is a

bead-based compensation tool specifically optimized for use with the Invitrogen™ LIVE/DEAD™

Fixable Dead Cell Stain Kits but also compatible with other assays that use amine-reactive

dyes. Established as a ready-to-use control for setting compensation on flow cytometers, the

ArC amine-reactive beads eliminate the hassle of heat-treating cells as a control, thus saving

your experimental samples while enabling accurate and consistent results. Learn more about

these compensation kits and the wide selection of fixable viability dyes for flow cytometry at

thermofisher.com/livedeadfixable.


ArC™ Amine-Reactive Compensation Bead Kit 1 kit, 25 tests

1 kit, 100 tests

A10628

A10346

New interleukin primary antibody conjugates for flow cytometryAntibodies validated for use in flow cytometry provide researchers with the ability to both identify

specific cell types and analyze the functional responses of heterogeneous cell populations. Part

of the cytokine family, interleukins are intracellular proteins produced by a variety of cell types

and used to study cell signaling. Our anti-interleukin antibodies now include brightly fluorescent

fluorescein, R-PE, and APC conjugates of anti–IL-27 antibodies for mouse and human cells.

Find more antibody conjugates for flow cytometry at thermofisher.com/flowantibodies.


Mouse Anti–Human IL-27 (Clone 307426), APC conjugate 100 tests MA5-23573

Mouse Anti–Human IL-27 (Clone 307426), fluorescein conjugate 100 tests MA5-23587

Mouse Anti–Human IL-27 (Clone 307426), R-PE conjugate 100 tests MA5-23686

Rat Anti–Mouse IL-27 (Clone 234205), APC conjugate 100 tests MA5-23603

Rat Anti–Mouse IL-27 (Clone 234205), fluorescein conjugate 100 tests MA5-23551

Rat Anti–Mouse IL-27 (Clone 234205), R-PE conjugate 100 tests MA5-23651

ArC kits for compensation of LIVE/DEAD Fixable Dead Cell Stains

Use of ArC beads to mimic staining with LIVE/

DEAD Fixable Dead Cell Stains. A mixture of positive

and negative beads from the ArC™ Amine-Reactive

Compensation Bead Kit was stained with LIVE/DEAD™

Fixable Violet Dead Cell Stain (Cat. No. L34955),

washed, and analyzed by flow cytometry using a

405 nm laser with 450/50 nm bandpass filter.

Specificity of a fluorescent anti–IL-27 conjugate.

Human peripheral blood mononuclear cells (PBMCs)

were stained with R-PE mouse anti–human IL-27 anti-

body (Cat. No. MA5-23686, orange) or isotype-control

antibody (blue) and analyzed by flow cytometry.

Western blot analysis of β-actin and β-tubulin in

five whole-cell lysates. Proteins transferred to a Low-

Fluorescence PVDF Membrane (Cat. No. 22860) were

probed with the Alexa Fluor™ 488 β-Actin (Cat. No.

MA1-140-A488) and Alexa Fluor™ 555 β-Tubulin (Cat.

No. MA5-16308-A555) Loading Control Antibodies.

IL-27 R-PE �uorescence

Num

ber

of c

ells

100 101 102 103 104

Eve

nts

coun

ted

0

100

200

300

400

LIVE/DEAD Fixable Violet stain �uorescence

10510410310210–2 0

130

β-Tubulinβ-Actin

1007055

35

25

15

kDa

HeL

a

U2O

S

CO

S-7

NR

K

NIH

/3T3

Alexa Fluor conjugates now available for epitope-tag and loading-control antibodies

http://thermofisher.com/tagabs

http://thermofisher.com/loadingctrlabs

http://thermofisher.com/livedeadfixable

http://thermofisher.com/flowantibodies


FEATUrED rEsEArCH TOOLs BIOPrOBEs 73

Optimize immunodetection with recombinant secondary antibodies

Superclonal secondary antibodies for sensitive and reproducible immunoassays.

Sensitive immunodetection in cells and tissues requires high-affinity antibodies that exhibit minimal

background staining. Invitrogen™ Superclonal™ secondary antibodies represent a breakthrough in

recombinant antibody technology, providing sensitive binding to their targets and very low levels of

nonspecific staining. Because of their consistent performance from lot to lot, these next-generation

secondary antibodies are becoming the reagent of choice in both research and clinical applications [1–4].

Figure 1 (above). Multicolor immunocytochemical analysis using fluorescent Superclonal secondary antibodies. U2OS (human osteocarcinoma) cells were labeled

with rabbit anti–acetyl-histone H3 (Lys9) primary antibody (ABfinity™ Rabbit Oligoclonal, clone 17HCLC, Cat. No. 710293) followed by the Alexa Fluor™ 647 conjugate of

Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody (Cat. No. A27040, red), and with mouse anti–α-tubulin primary antibody (clone B-5-1-2, Cat. No. 32-2500)

followed by the Alexa Fluor™ 488 conjugate of Goat Anti–Mouse IgG (H+L) Superclonal™ Secondary Antibody (Cat. No. A28175, green).


BIOPrOBEs 73 FEATUrED rEsEArCH TOOLs

Advantages of Superclonal recombinant antibodies over traditional polyclonalsSuperclonal recombinant secondary antibodies enable the accurate

and precise detection of mouse, rabbit, and goat IgG antibodies in cell

imaging (Figure 1), flow cytometry, western blot, and ELISA applica-

tions, with little to no cross-reactivity to IgGs from other species. Their

recombinant origin helps ensure consistency between lots, minimizing

the need to optimize each lot before using them in an immunoassay.

By comparison, typical polyclonal secondary antibodies are affinity

purified from the serum of immunized animals, resulting in a large,

undefined pool of antibodies with unknown epitope-binding charac-

teristics. Although broad epitope coverage is a benefit of traditionally

produced polyclonal secondary antibodies, poor lot-to-lot consistency

due to animal variability and purification processes can lead to sig-

nificant cross-reactivity and high background signals. With extensive

validation using a panel of primary antibodies, we have demonstrated

that Superclonal secondary antibodies show high affinity to their

target IgG species with minimal cross-species reactivity, providing

consistently high signal-to-noise ratios across lots and in a variety of

immunodetection protocols.

Producing high-quality recombinant antibodiesTo produce Superclonal secondary antibodies, we employ recombinant

technology followed by a multifaceted clonal selection process that

involves several phenotypic screens (Figure 2). First we construct a

cDNA library of IgG light chain and heavy chain genes from goat and

rabbit peripheral blood mononuclear cells (PBMCs), and then reverse

engineer these genes for heterologous co-expression [5]. Library

construction is followed by thorough screening and selection of the

monoclonal antibodies. Throughout the screening process, we use

highly cross-adsorbed polyclonals as a benchmark to eliminate clones

with similar or higher cross-reactivity to IgGs from closely related

Figure 2. Superclonal secondary antibodies—a defined pool of well-characterized monoclonal antibodies. The development of a Superclonal™ secondary antibody

entails the construction of IgG light chain (LC) and IgG heavy chain (HC) cDNA libraries using goat and rabbit peripheral blood mononuclear cells (PBMCs), followed by com-

binatorial screens to produce multiple monoclonal antibody candidates (mAbs). The mAbs are then analyzed for affinity as well as performance in several immunodetection

protocols (ELISA, western blotting, and immunocytochemical (ICC) assays); the corresponding LC and HC cDNAs are also sequenced to confirm diversity among the individual

mAbs. The mAbs that exhibit superior performance in the immunoassays and show little or no cross-reactivity are pooled to produce a Superclonal secondary antibody.

Characterize monoclonals

Analyze

Pool

Affi nity assay ELISA ICC cDNA sequenceWestern

Isolate PBMCs (goat and rabbit)

Construct IgG cDNA libraries

Screen monoclonals

Select multiple monoclonals Superclonal

IgG LC cDNA+

IgG HC cDNA


FEATUrED rEsEArCH TOOLs BIOPrOBEs 73

species. Finally, monoclonals are selected

based on their high affinity to the target IgG

species along with specificity equal to or

better than that of the polyclonal benchmarks

across a variety of immunodetection proto-

cols, including cell imaging, flow cytometry,

western blot, and ELISA applications.

Selected monoclonal antibodies with high

sensitivity and specificity are then sequenced

and pooled to simulate the diversity of a poly-

clonal antibody. Several iterations of this pool

are tested to find the combination that yields

the highest signal-to-noise ratio in various

immunoassays. The resulting “superclonal”

mixture of recombinant goat or rabbit second-

ary antibodies binds with the epitope-specific

precision of monoclonal antibodies, while

also achieving the multi-epitope coverage

(e.g., toward both heavy and light chains of

target IgGs) and signal amplification of poly-

clonal antibodies. These well-characterized

Superclonal secondary antibodies are then

conjugated to brightly fluorescent Invitrogen™

Alexa Fluor™ dyes (Figure 3), or to horse-

radish peroxidase (HRP) or biotin, and sub-

jected to stringent testing to confirm their

performance across detection platforms. In

direct ELISAs, the Superclonal secondary

antibodies show little or no cross-reactivity

with IgGs from other species, whereas the

highly cross-adsorbed polyclonals show

dose-dependent cross-reactivity to IgGs from

closely related species (Figure 4). Furthermore,

Figure 4. Comparison of IgG species cross-reactivity. To compare the cross-reactivity of Superclonal™

secondary antibodies to highly-cross adsorbed polyclonal secondary antibodies, colorimetric ELISAs using purified

goat, guinea pig, bovine, and mouse IgGs were performed. (A) When comparing rabbit anti–goat IgG secondary

antibodies (RAG), the polyclonal antibody showed a dose-dependent cross-reactivity to bovine IgG, whereas the

Superclonal antibody (Cat. No. A27011) did not cross-react with any of the species tested. (B) When comparing

rabbit anti–mouse IgG secondary antibodies (RAM), the two polyclonal antibodies tested cross-reacted with rat

IgG (polyclonal 1) or both goat and rat IgG (polyclonal 2), whereas the Superclonal antibody (Cat. No. A27022)

displayed minimal cross-reactivity.

Figure 3. Multiplex immunocytochemical analysis using fluorescent Superclonal secondary

antibodies. SH-SY5Y (human neuroblastoma) cells were labeled with mouse anti-Aβ40 primary

antibody followed by the Alexa Fluor™ 488 conjugate of Goat Anti–Mouse IgG (H+L) Superclonal™

Secondary Antibody (Cat. No. A28175, green), and with rabbit anti-DISC1 primary antibody

(ABfinity™ Rabbit Oligoclonal, Cat. No. 710203) followed by the Alexa Fluor™ 555 conjugate of

Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody (Cat. No. A27039, red). Nuclei

were stained with SlowFade™ Gold Antifade Mountant with DAPI (Cat. No. S36938, blue). Images

were captured at 60x magnification.

0

1.0

2.0

3.0

Ab

sorb

ance

(450

nm

)

300.

0010

0.00

33.3

311

.11

3.70

1.23

0.41

0.14

0.05

0.02

Concentration (ng/mL)

Mouse IgG

Bovine IgG

0

1.0

2.0

3.0

Ab

sorb

ance

(450

nm

)

Guinea pig IgG

Ab

sorb

ance

(450

nm

)

0

1.0

2.0

3.0

Ab

sorb

ance

(450

nm

)

0

1.0

2.0

3.0Superclonal RAGPolyclonal RAG

Goat IgG

Bovi

neRat

Mou

se Pig

Hor

se

Donk

ey

Hum

an

Goa

t

Ab

sorb

ance

(450

nm

)

0

1.0

2.0

3.0Polyclonal 2 RAM

Ab

sorb

ance

(450

nm

)

0

1.0

2.0

3.0

Polyclonal 1 RAM

Ab

sorb

ance

(450

nm

)

1,370 ng274 ng

55 ng

0

1.0

2.0

3.0

Superclonal RAMBA


BIOPrOBEs 73 FEATUrED rEsEArCH TOOLs

Superclonal secondary antibodies exhibit much lower background signals

in immunocytochemical assays (Figure 5). To date we have validated

the Superclonal secondary antibody conjugates with more than 300

primary antibodies for immunodetection applications.

Consistent performance across lotsTo ensure reproducible performance of each of the Superclonal

antibodies, we run quantitative analyses for every unconjugated and

conjugated antibody. Figure 6 shows the performance of five different

Selected Superclonal secondary antibody products Quantity Cat. No.

Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor™ 488 conjugate

1 mg A27034

Goat Anti–Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor™ 488 conjugate

1 mg A28175

Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate

1 mg A27036

Goat Anti–Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate

1 mg A28177

References1. Bradbury A, Plückthun A (2015) Nature 518:27–29.

2. Bradbury AM, Plückthun A (2015) Nature 520:295.

3. Karn AE, Bell CW, Chin TF (1995) ILAR J 37:132–141.

4. Wang M, Wang Y, Zhong J (2015) Mol Med Rep 11:4297–4302.

5. Seeber S, Ros F, Thorey I et al. (2014) PLoS One 9:e86184.

Figure 6. Comparison of Superclonal secondary antibody performance across

multiple lots. Western blot analysis was performed on whole cell extracts (20 µg

lysate) of HeLa (human cervical carcinoma) cells for detection of (A) endogenous

α-Akt (~60 kDa) and (B) endogenous α-Traff (~63 kDa), using the corresponding

rabbit primary antibodies. Each lane on the blots was then incubated with one of

five different lots of Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody

(Cat. No. A27033), followed by the HRP conjugate of Rabbit Anti–Goat IgG (H+L)

Superclonal™ Secondary Antibody (Cat. No. A27014); the last lane was incubated

with primary antibody and the benchmark polyclonal secondary antibody (pAb).

Endogenous tubulin, detected with mouse anti-tubulin primary antibody and anti–

mouse IgG secondary antibody, was used as a control for loading.

Figure 5. Reduction in nonspecific staining with Superclonal secondary anti-

bodies. Nucleoli of HeLa (human cervical carcinoma) cells were labeled with mouse

anti-nucleostemin primary antibody, which was then detected with the Alexa Fluor™

488 conjugate (green) of (A) highly cross-adsorbed goat anti–mouse IgG polyclonal

antibody or (B) Goat Anti–Mouse IgG (H+L) Superclonal™ Secondary Antibody

(Cat. No. A28175). Similar intensity of staining in the nucleolus is observed with

both secondary antibodies, but the Superclonal antibody exhibits significantly less

cytoplasmic staining, indicating enhanced specificity.

B

A

Loading control

α-Traff

1520

30

4050

60

260

16011080

1 2 3 4 5pAbkDa

kDa

Loading control

α-Akt

1520

30

4050

60

260

16011080

Lot number1 2 3 4 5

pAb

lots of unconjugated Goat Anti–Rabbit IgG (H+L) Superclonal Secondary

Antibody on western blots. We routinely compare the performance of

newly manufactured lots of a Superclonal antibody to previous lots using

several different immunoassay formats (cell imaging, flow cytometry,

western blot, ELISA) and quantify the results with standard densitometry

or fluorescence-based algorithms. In all cases, Superclonal secondary

antibodies show consistent performance across lots, unlike currently

available polyclonal secondary antibodies, which require optimization

by the researcher for each new lot due to variability that can arise

from the animal sources and purification procedures. The lot-to-lot

consistency of Superclonal antibodies minimizes the need to optimize

each lot before using it in an immunodetection protocol, not only saving

time but producing results that are reproducible from day to day and

from experiment to experiment.

See the entire line of Superclonal secondary antibodiesSuperclonal secondary antibodies are available unconjugated, as well

as conjugated to biotin, HRP, and the brightly fluorescent Alexa Fluor

dyes. See our complete selection of Superclonal secondary antibodies

at thermofisher.com/superclonalbp73. ■

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bradbury%20A%5BAuthor%5D&cauthor=true&cauthor_uid=25652980

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pl%C3%BCckthun%20A%5BAuthor%5D&cauthor=true&cauthor_uid=25652980

http://www.ncbi.nlm.nih.gov/pubmed/25877194

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bradbury%20AM%5BAuthor%5D&cauthor=true&cauthor_uid=25877194

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pl%C3%BCckthun%20A%5BAuthor%5D&cauthor=true&cauthor_uid=25877194

http://www.ncbi.nlm.nih.gov/pubmed/25877194

http://www.ncbi.nlm.nih.gov/pubmed/?term=Seeber%20S%5BAuthor%5D&cauthor=true&cauthor_uid=24503933

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ros%20F%5BAuthor%5D&cauthor=true&cauthor_uid=24503933

http://www.ncbi.nlm.nih.gov/pubmed/?term=Thorey%20I%5BAuthor%5D&cauthor=true&cauthor_uid=24503933

http://www.ncbi.nlm.nih.gov/pubmed/?term=A+Robust+High+Throughput+Platform+to+Generate+Functional+Recombinant+Monoclonal+Antibodies+Using+Rabbit+B+Cells+from+Peripheral+Blood

https://www.thermofisher.com/order/catalog/product/A28175

http://thermofisher.com/superclonalbp73


PrOTEIN FUNCTION AND ANALYsEs BIOPrOBEs 73

Immune cell surveillance by an array of cell

types not only ensures the maintenance

of tissue structure and function but also is

responsible for mounting acute inflammatory

responses that protect the host from patho-

gens. During an insult resulting from infection

or trauma, granulocytes are recruited to sites

of acute inflammation to confine tissue dam-

age and initiate healing. Leukocytes, such as

macrophages and other cell types, migrate

from the peripheral circulation to sites of

injury to remove damaged cells and debris,

which facilitates resolution of inflammation.

Finally, fibroblasts are required for extracellular

matrix deposition and vascular endothelial

cells mediate angiogenesis—both processes

required for tissue repair.

A large body of literature has reported

that malignancy is frequently associated with

chronic, persistent, pathological inflammation

[1–3]. Moreover, with respect to cancer pro-

gression, several investigations have revealed

that specific populations of immune cells

possess either anti-tumorigenic properties or

the ability to promote tumorigenesis. Disease

progression may therefore depend on the

balance of these cell subsets and the milieu

of soluble mediators.

We recent ly compi led a handbook

of ant ibody-based tools for evaluat ing

tumor-related inflammation (download it at

thermofisher.com/tumorinflammation-hb).

Table 1 (excerpted from this handbook)

provides an overview of proinflammatory

proteins associated with leukocytes in the

tumor microenvironment, along with anti-

body-based tools for a variety of experimental

applications.

Cells and soluble mediators of the tumor microenvironmentAntibody-based tools for identifying cell types and proteins of interest.

Figure 1. Representation of cell components of the inflammatory tumor microenvironment. Cells that

reside within solid tumors—including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells

(MDSCs), T regulatory cells (Tregs), and other cells—actively suppress antigen-presenting cells such as dendritic

cells and inhibit productive anti-tumor responses mediated by cytotoxic CD8+ T cells (CTLs) and natural killer cells.

Dissecting the role of different cell types in tumor-related inflammationThe inflammatory tumor microenvironment comprises a wide variety of cell types (Figure 1).

Recent studies of cancer stroma and immune system–based biomarker profiles in solid tumors

indicate that survival or response to cancer therapy may either be positively or negatively

correlated with the ratios of specific cell populations. For example, research studies of human

breast cancer demonstrated that stromal cancer-associated fibroblasts (CAFs) contribute to

the tumorigenic microenvironment and are negatively correlated with survival. These cells pro-

duce the chemokine stromal cell–derived factor 1 (SDF-1, also known as CXCL12)—a strong

lymphocyte and macrophage chemoattractant—which signals through the receptor CXCR4 to

promote tumor cell growth, invasion, and tumor angiogenesis [4–6].

Published reports have established that the presence of macrophages in a number of

different cancers correlates with increased vascular density and a poor clinical outcome, and

the presence of tumor-associated macrophages (TAMs) supports tumor growth. Furthermore, a

cell signature consisting of a high level of macrophages and CD4+ T cells relative to low levels

of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) was predictive of reduced survival.

In addition, various studies employing mouse models suggest that manipulation of the tumor

microenvironment by depletion of mammary TAMs—cells that promote inflammation and inhibit

tumoricidal responses—may enhance the response to chemotherapy and augment the activity

of tumor-specific CTLs [5].

The field of tumor immunology recently realized great successes with the Food and Drug

Administration (FDA) approval of three novel biotherapeutic agents designed to boost CTL-

mediated rejection of solid tumors. Years of work in cell immunology and cancer research

enabled the development of ipilimumab, an anti–CTLA-4 monoclonal antibody for the treatment

T cell subsets

• CD8

• CD4

– Th1 and Th2 helper cells– Th17 cells– Tregs

Red blood cell

Malignant cell

Necrotic or hypoxic malignant cell

Dendritic cell

Natural killer cell

B cell

Fibroblast

Endothelial cell

Myeloid cell subsets

• Tumor-associated macrophage (TAM)

• Myeloid-derived suppressor cell (MDSC)

http://thermofisher.com/tumorinflammation-hb


BIOPrOBEs 73 PrOTEIN FUNCTION AND ANALYsEs

of melanoma, and pembrolizumab and nivolumab, two anti–PD-1

monoclonal antibodies for the treatment of metastatic melanoma and

metastatic non-small cell lung carcinoma (NSCLC), respectively. New

strategies designed to enhance anti-tumor immune responses and

reduce pro-tumorigenic conditions are under intense investigation [7,8].

Antibody-based analysis of the tumor microenvironmentThe role of antibodies in basic and translational research cannot

be overstated. The ability to produce highly purified target-specific

antibodies has made it possible to detect, quantify, and observe

the ways in which specific proteins function within tissue, cells, and

extracellular compartments. The use of research antibodies has an

important place in the greater context of powerful preclinical cancer

models, genomics and proteomics tools, and cellular imaging modal-

ities that continue to fuel advances in basic science and medicine.

Characterization of cell populations that frequently infiltrate solid

tumors relies, in large part, on the ability to perform these procedures:

(1) liberate cells from solid tumors for analysis by flow cytometry;

(2) detect cells in tissues by histology, immunohistochemistry, and

immunofluorescence; and (3) detect signature cytokines and growth

factors linked to tumor-related inflammation using various soluble-protein

detection methods such as enzyme-linked immunosorbent assays

(ELISAs) and western blots. Table 1 provides an overview of key fea-

tures attributed to cell populations commonly associated with the tumor

microenvironment, and provides a list of representative antibodies and

ELISA kits that may be used for protein and cell analysis in a range

of applications. In our handbook Antibody-based tools for evaluating

References1. Coussens LM, Werb Z (2002) Nature 420:860–867.

2. Shiao SL, Ganesan AP, Rugo HS et al. (2011) Genes Dev 25:2559–2572.

3. Medzhitov R (2008) Nature 454:428–435.

4. DeNardo DG, Brennan DJ, Rexhepaj E et al. (2011) Cancer Discov 1(1):54–67.

5. Gajewski TF, Schreiber H, Fu YX (2013) Nat Immunol 14:1014–1022.

6. Yang L, Karin M (2014) Cell Death Differ 21:1677–1686.

7. National Cancer Institute. FDA Approval for Ipilimumab. http://www.cancer.gov/about-cancer/treatment/drugs/fda-ipilimumab. Accessed March 2015.

8. FDA News Release (October 2, 2015). FDA approves Keytruda for advanced non-small cell lung cancer. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm465444.htm.

Table 1. Proinflammatory proteins associated with leukocytes of the tumor microenvironment.

Protein Source Antibody Cat. No. ELISA Cat. No. Functions in linking inflammation to cancer

IL-6 MDSCs, TAMs 700480 KHC0061 • Promotes tumor growth

IL-8 MDSCs, TAMs 710256 KHC0081 • Enhances proliferation, migration, and angiogenesis

IL-10 MDSCs, TAMs, Tregs 710170 KHC0101• Anti-inflammatory• Downregulates expression of Th1 cytokines and co-stimulatory

molecules on macrophages

IL-17 Th17 cells PA1-84183 EHIL17A • Associated with angiogenesis and poor prognosis

IL-21 Th17 cells 710141 EHIL21 • Associated with tumor development

IL-22 Th17 cells 701031 EHIL22 • Promotes differentiation of Th17 cells

TNF-αMacrophages, monocytes, neutrophils, T cells, NK cells

710288 KHC3011• Induces DNA damage and inhibits DNA repair• Promotes tumor growth• Induces angiogenic factors

GM-CSFMacrophages, T cells, mast cells, NK cells, endothelial cells, fibroblasts

701136 KHC2011 • Activates macrophages

M-CSF Endothelial cells, fibroblasts PA1-20182 EHCSF1 • Controls production, differentiation, and function of macrophages

tumor-related inflammation, we provide links to antibody-based tools

as well as access to detailed protocols for a number of experimental

techniques, including western blot, ELISA, immunofluorescence analysis,

flow cytometry, and more.

Access our antibody resources todayFind out more about this growing area of research by downloading our

latest handbook, Antibody-based tools for evaluating tumor-related

inflammation, at thermofisher.com/tumorinflammation-hb. We also

offer other downloadable handbooks that describe cancer signaling

pathways, stem cells, and neurodegenerative disease research, as well

as the related antibody-based resources, all of which can be accessed

by completing the form at thermofisher.com/abtoolshandbooks. To

explore our primary and secondary antibody search tool and learn more

about immunoassays (including Invitrogen™ multi-analyte assays for the

Luminex™ platform), antibody labeling, and other antibody applications,

visit thermofisher.com/antibodybp73. ■

http://www.cancer.gov/

http://thermofisher.com/tumorinflammation-hb

http://thermofisher.com/abtoolshandbooks

http://thermofisher.com/antibodybp73



The study of complex cellular signaling pathways requires powerful,

specific tools to accurately monitor changes in gene activation or repres-

sion. Because of their ultrasensitive detection capabilities and wide

dynamic range, luciferase-based reporter gene assays are widely used

to measure the activity of promoters and other transcriptional regulatory

elements, as well as the effects of activators and inhibitors. However,

the “flash” type enzyme kinetics associated with the conventional

ultrasensitive luciferase assay format requires specialized equipment

for measurement; and the enzymatic nature of the reporter makes

these assays susceptible to modulation by a variety of small-molecule

compounds found in screening collections, preventing their adoption

in high-throughput screening (HTS) applications.

Meet the innovative TurboLuc luciferaseThe Thermo Scientific™ TurboLuc™ Luciferase One-Step Glow Assay

was designed to measure luciferase activity in mammalian cells with

the addition of a single reagent, making it ideal for HTS applications.

This one-step homogeneous assay employs a novel 16 kDa ATP-

independent luciferase (TurboLuc16, or Tluc16)—the smallest luciferase

described to date—derived from a marine copepod of the genus

Metridia. The wild-type luciferase was modified to reduce its size,

increase its brightness, and enable its efficient intracellular expression

(Figure 1A). The TurboLuc gene was further modified to include patented

dual-destabilization elements that reduce nonspecific accumulation

of the TurboLuc mRNA and protein in cells (Figure 1B), enhancing

the responsiveness and sensitivity of the assay (Figure 1C). With the

addition of coelenterazine, Tluc16 produces intense blue-luminescent

light that is stable over the detection period, enabling measurement of

very minute amounts of luciferase activity using a standard luminometer,

HTS instrument, or other automated detection platform.

Robust performance in high-throughput formatsWhen compared with the Promega NanoLuc™ system, the TurboLuc

Luciferase One-Step Glow Assay shows brighter signals (Figure 2A), as

well as a similarly stable signal over time (Figure 2B). With its increased

luminescence response, stable glow kinetics, and simple one-step

protocol, the TurboLuc luciferase assay has been specifically designed

for use in HTS applications and other laboratory automation formats

and should prove especially beneficial when detecting low or tran-

sient levels of gene expression. Figure 3 demonstrates the use of the

Figure 1. Optimizing the TurboLuc16 luciferase for high-throughput screening applications. (A) Mutagenesis of the wild-type luciferase from Metridia has produced a

luciferase reporter enzyme with desirable glow kinetics. HEK 293 cells were transfected with plasmids containing the dual-destabilized TurboLuc16 luciferase (Tluc16-DD)

gene or the non-optimized luciferase gene, and luciferase activity was measured using the TurboLuc Luciferase One-Step Glow Assay Kit over a 1 hr period. (B) The presence

of dual-destabilization (DD) elements reduces accumulation of luciferase in cells. Luciferase activity was measured in HEK 293 cells transfected with plasmids containing

either the dual-destabilized Tluc16 gene (pMCS minP-Tluc16-DD, Cat. No. 88232) or the nondestabilized Tluc16 gene (pMCS minP-Tluc16, Cat. No. 88236) under the

control of an optimized minimal core promoter (minP) designed for the measurement of nonspecific expression of the luciferase reporters. (C) The TurboLuc16 luciferase

with dual-destabilization technology shows improved responses in biological assays. Luciferase activity was measured in HEK 293 cells transfected with plasmids containing

either the dual-destabilized Tluc16 gene (pCRE-Tluc16-DD, Cat. No. 88247) or the nondestabilized Tluc16 gene (pCRE-Tluc16) under the control of a combination of an

optimized minimal core promoter and five tandem repeats of the cAMP response element (CRE). Results are displayed as fold induction of Tluc16 activity in cells treated

with 10 µM forskolin (to raise cAMP levels) relative to untreated cells.

Monitor changes in gene expression using luminescenceThe HTS-compatible TurboLuc Luciferase One-Step Glow Assay.

Fold

cha

nge

in a

ctiv

ity

afte

r in

duc

tion

pCRE Tluc16

pCRE Tluc16-DD

50

0

100

150

200

250

300

350

400

20,000

30,000

40,000

50,000

60,000

pMCS minP-Tluc16

pMCS minP-Tluc16-DD

10,000

0

Rel

ativ

e lu

min

esce

nce

units

(RLU

)

40320 48 5624168

Time after reagent addition (min)

Non-optimized TlucTluc16

60,000

0

20,000

40,000

80,000

100,000

Rel

ativ

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min

esce

nce

units

(R

LU)

A B C



Figure 2. Comparing the response and glow kinetics (signal stability) of the TurboLuc and NanoLuc

reporter systems. HEK 293 cells were transfected with either a CRE or NF-κB transcriptional reporter plasmid

containing either the dual-destabilized TurboLuc16 luciferase gene (Tluc16-DD) or the destabilized Promega

NanoLuc™ luciferase gene (NlucP), and luciferase activity was measured using the recommended assay reagents.

(A) Luciferase activity is shown for the Tluc16-DD and NlucP reporter systems as fold induction in luciferase activity

in cells treated with either 10 µM forskolin (for CRE plasmids) or 10 ng/mL TNF-α (for NF-κB plasmids) relative to

untreated cells. (B) Glow kinetics (signal stability) of the Tluc16-DD and NlucP reporter systems (using the CRE

plasmids) was compared by recording the luminescence signal emitted by these luciferases over a 1 hr period.

Figure 3. High-throughput screening with the

TurboLuc luciferase assay: Performance over

several different cell concentrations. After trans-

fection with a plasmid containing the Tluc16-DD

gene under the control of NF-κB response elements

(pNF-κB Tluc16-DD, Cat. No. 88246), HEK 293 cells

were plated into multiple 384-well plates and then

stimulated with a series of TNF-α concentrations to

generate dose-response curves at different cell con-

centrations. On average, Z´ values for these assays

were greater than 0.7.


TurboLuc™ Luciferase One-Step Glow Assay Kit 100 reactions1,000 reactions

8826388264

pMCS minP-Tluc16-DD Vector for Luciferase Assays 10 µg 88232

pMCS minP-Tluc16 Vector for Luciferase Assays 10 µg 88236

pCRE Tluc16-DD Vector for Luciferase Assays 10 µg 88247

pNF-κB Tluc16-DD Vector for Luciferase Assays 10 µg 88246

BA

Time after reagent addition (min)

0 705030 604020 80

Rel

ativ

e lu

min

esce

nce

units

(R

LU)

Tluc16-DDNlucP

Fold

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in a

ctiv

ity

afte

r in

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tion

NF-κB plasmidCRE plasmid0

40

80

160

100

1,000

10,000

100,000

120

Tluc16-DDNlucP

log [TNF-α] (ng/mL)–3 1–1–2 20

Rel

ativ

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esce

nce

units

(RLU

)

25,000

50,000

75,000

100,00010K cells/well

5K cells/well

2K cells/well

1K cells/well

TurboLuc assay to generate dose-response

curves using an HTS platform. After transfec-

tion with a plasmid containing the Tluc16-DD

gene under the control of NF-κB response

elements, HEK 293 cells were plated into

multiple 384-well plates and then stimulated

with a series of TNF-α concentrations to gen-

erate dose-response curves at different cell

numbers. The luminescence response was

reproducibly measured over several orders of

magnitude in TNF-α concentration and one

order of magnitude in cell concentration. The

combination of brightness, responsiveness,

and stable glow characteristics makes the

TurboLuc system an ideal platform for reporter

gene assays for HTS applications.

TurboLuc expression vectors: Choose from a variety of transcriptional regulation elementsThe Tluc16 luciferase expression vectors are

offered in a variety of configurations to enable

different experimental workflows. All of these

cloning vectors contain unique multiple cloning sites (MCS) to accept transcriptional regulators

such as promoters or response elements. The minP-Tluc16 vectors contain a core minimal

promoter (minP) designed for studying the regulation of transcription by elements lacking pro-

moter activity. The Tluc16-DD expression vectors incorporate the patented dual-destabilization

(DD) technology. To minimize assay variability, stable cell lines selected for genetically homo-

geneous expression are desirable. These stable cell lines can be generated either through

positive selection using Tluc16 vectors containing the gene for hygromycin resistance or through

the use of cell engineering tools such as the BacMam gene delivery and expression system,

Invitrogen™ Jump-In™ technology, or other gene-editing methods.

Learn about the TurboLuc Luciferase One-Step Glow AssayThe TurboLuc™ Luciferase One-Step Glow Assay Kit contains an assay buffer and substrate

solution that have been specifically developed to function in a one-step homogeneous assay

format that is amenable to laboratory automation methods. Find out more about the TurboLuc

Luciferase One-Step Glow Assay, as well as our selection of over 10 different Tluc16 expression

vectors, at thermofisher.com/turbolucbp73. ■

http://thermofisher.com/turbolucbp73



Cast your protein gels with easeIntroducing the SureCast Gel Handcast System for protein separation.

Figure 2. SureCast Gel Handcast System’s leak-free design. (A) Gel sandwich

using a single U-shaped spacer, from the SureCast™ Gel Handcast System.

(B) Load-and-lock mechanism of the SureCast Gel Handcast System.

Polyacrylamide gel electrophoresis (PAGE)—an important technique for

separating proteins based on their electrophoretic mobility—is widely

used for both protein analysis and protein purification. To prepare a

polyacrylamide gel, polymerization catalysts (e.g., TEMED and APS)

are added to a solution of acrylamide/bisacrylamide, which is imme-

diately poured between two glass plates separated by spacers and

subsequently polymerizes into a solid gel. One major challenge in

casting these gels is leakage of this toxic acrylamide solution out of

the glass-plate sandwich before polymerization occurs. This leakage

can be caused by multiple factors, including worn spacers, uneven

pressure in the gel holder, and breakage of the glass plates.

The Invitrogen™ SureCast™ Gel Handcast System (Figure 1) features

innovative design elements that make casting protein gels easier and

more efficient than ever before. These design elements include:

■ A leak-free* design, which minimizes the number of failed gels

and wastes less time

■ Simple component assembly that uses a single-motion load-and-

lock mechanism

■ A unique tilt feature, which minimizes spillage during pouring of

acrylamide solutions

■ Superior glass plates, specifically developed to maximize durability

Unique leak-free designOne problem with current protein gel casting systems is the use of

two to three separate spacers in the glass-plate sandwich. If these

spacers are not placed in exactly the right positions on the plates,

the seal of the sandwich can be compromised and the gel solution

will leak out before it has time to polymerize. The SureCast Gel

Handcast System uses a single U-shaped spacer (Figure 2A) that

provides a continuous barrier around the bottom and sides of the

plates, allowing it to be placed in the correct position every time. The

SureCast system has been designed to be 100% leak free throughout

the casting process.

Additionally, the SureCast Gel Handcast Station features an inno-

vative load-and-lock mechanism that securely seals the glass plates

by evenly distributing force all along the sealing edges (Figure 2B), in

contrast to other commercially available chambers that provide force

only on the sides of the plates. The SureCast Gel Handcast Station

also stabilizes the gel assembly while the acrylamide solution is poured,

with the added benefit of reducing the formation of bubbles.

SureCast™ Glass Plates are unlike any glass plates used in casting

systems today. These plates are thicker and more damage-resistant

than other manufacturers’ plates, enabling more gels to be cast and

run over the lifetime of the plates. However, it is not only the strength

of the glass plates that contributes to the system’s leak-free abilities:

the sealing area is specifically designed to minimize the effects of

any damage to the edges of the glass plates. When coupled with the

even-sealing mechanism of the SureCast Gel Handcast Station, these

durable glass plates ensure that minor cracks or chips will not impact

the sealing ability of the SureCast system.

BA

Figure 1. The complete SureCast Gel Handcast System. The SureCast™ Gel

Handcast System is available in convenient hardware bundles—including casting

stations, glass plates, spacers, and multi-use tools—with and without reagents.




Hardware and bundles

SureCast™ Gel Handcast Bundle A (Hardware and Reagents) 1 kit HC1000SR

SureCast™ Gel Handcast Bundle B (Hardware Only) 1 kit HC1000S

SureCast™ Gel Handcast Station 1 station HC1000

SureCast™ Gel Spacer 10 spacers HC1003

SureCast™ Glass Plates 2 sets HC1001

SureCast™ Sealing Pads 2 pads HC1002

SureCast™ Multi-Use Tool, 10-well 1 unit HC1010



Reagents

SureCast™ Acrylamide Solution (40%) 450 mL HC2040

SureCast™ TEMED 30 mL HC2006

SureCast™ APS 25 g HC2005

SureCast™ Resolving Buffer, dry powder 2 x 500 mL HC2212

SureCast™ Resolving Buffer, dry powder 5 x 500 mL HC2215

SureCast™ Stacking Buffer, dry powder 2 x 500 mL HC2112

SureCast™ Stacking Buffer, dry powder 5 x 500 mL HC2115

Choose the right systemThe complete SureCast Gel Handcast System

includes the hardware along with a set of SureCast

reagents that offer superior storage, safety, and

convenience when compared with other com-

mercially available solutions. Also available sep-

arately, these SureCast reagents have long shelf

lives (up to 2 years) and can be safely stored at

room temperature. Convenient starter and combo

bundles containing all hardware and reagents

are available. Find out more about our SureCast

Gel Handcast System and related products at

thermofisher.com/surecastbp73. ■

* Terms and conditions apply;

visit thermofisher.com/surecast-leakfree-terms

for more information.

Load more sample with these precast protein gelsNovex WedgeWell Tris-glycine precast protein gels.

Today many labs depend on the convenience and consistency provided by

commercially available, ready-to-use, precast protein gels. Technological

advances in buffer formulations and gel polymerization methods have enabled

manufacturers to produce precast gels—even gradient gels—with greater

uniformity than ever before. Moreover, the use of precast gels means less

exposure to the acrylamide monomer (a known neurotoxin and suspected

carcinogen) for the bench scientist.

The next generation of precast protein gelsBased on traditional Laemmli protein electrophoresis, the Invitrogen™

Novex™ WedgeWell™ precast protein gels (Figure 1) include innovations in

both chemistry and design that produce:

■ Improved shelf life—store gels for up to 12 months at 4°C

■ Increased sample capacity—easily load up to 60 µL of sample per well

■ Reliable protein detection—obtain exceptional protein band resolution

and sharpness

■ Fast run conditions—quickly separate proteins using constant voltage

in less than 60 minutes

■ More flexibility in experimental design—gels can be used to run native

or denatured protein samples

Extended shelf lifeOne common drawback with precast gels is their relatively short

shelf life, which typically ranges from four to eight weeks after

purchase. Novex WedgeWell precast protein gels have been

designed to minimize the effects of polyacrylamide hydrolysis at

the high pH of a typical Tris-glycine resolving gel, which results in

a shelf life of up to 12 months when stored at 4°C.

Figure 1. The Novex WedgeWell precast protein gel system. Each

Novex™ WedgeWell™ Welcome Pack includes a Mini Gel Tank, two boxes

of Novex WedgeWell precast gels (20 total), sample and running buffers,

reducing agent, and two vials of PageRuler™ Plus Prestained Protein Ladder

(10 to 250 kDa).

http://www.thermofisher.com/order/catalog/product/HC1000SR

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A B20 µL 30 µL 40 µL 50 µL 60 µL 20 µL 30 µL 40 µL 50 µL 60 µL

WedgeWell Tris-glycine 10-well gel Supplier B 10-well gel

for transfer of larger amounts of protein to western blots and more

sensitive detection of low-abundance proteins.

Improved band resolution and flexibilityThe modified chemistry of the Novex WedgeWell precast protein gels

produces less band distortion and greater band resolution, as com-

pared with classic Laemmli gels used by other suppliers (Figure 3).

Additionally, Novex WedgeWell gels do not contain SDS and can

therefore be used to run proteins in either native or denatured form,

using either the Invitrogen™ Novex™ Tris-Glycine Native Sample Buffer

and Running Buffer or the Invitrogen™ Novex™ Tris-Glycine SDS Sample

Buffer and Running Buffer.

Getting started is easyWe offer a variety of Novex WedgeWell Welcome Packs that include the

most popular Tris-glycine products needed to get started, as well as an

online selection guide to help you choose the right Novex WedgeWell

product for your lab. For these tools and a complete product list, visit

thermofisher.com/novexwedgebp73. ■

Figure 2. Increased sample volume capacity of Novex WedgeWell Tris-glycine gels. (A) To compare gel spillover, increasing volumes (20–60 μL) of a fluorescent

protein ladder were loaded in every other lane of a Novex™ WedgeWell™ Tris-glycine 10-well gel (left) or another supplier’s gel (right). Sample spillover in Supplier B’s gel is

seen in lanes adjacent to the 50 μL and 60 μL loading lanes. (B) Close-up view of the proprietary wedge-shaped well design of the Novex WedgeWell precast protein gels.

Figure 3. Superior band quality with Novex WedgeWell Tris-glycine gels.

Protein ladders, purified proteins, and E. coli lysate were loaded on (A) a Novex™

WedgeWell™ 4–20% Tris-Glycine Gel (Cat. No. XP04200PK2) and (B) another

supplier’s 4–20% gradient gel. Lanes 1, 5, 10: 5 µL PageRuler™ Unstained Protein

Ladder (Cat. No. 26614); lanes 2, 6, 9: 5 µL Mark12™ Unstained Standard (Cat.

No. LC5677); lane 3: 10 µL 1 mg/mL E. coli lysate; lane 4: 10 µL 0.6 mg/mL

BSA; lane 7: 10 µL 0.6 mg/mL Human IgG (Cat. No. 31154); lane 8: 20 µL

1 mg/mL E. coli lysate.

Selected Novex WedgeWell products Quantity Cat. No.

Novex™ WedgeWell™ Welcome Pack, 10%, 10-well 1 kit XP0010A

Novex™ WedgeWell™ Welcome Pack, 10%, 15-well 1 kit XP0010C

Novex™ WedgeWell™ Welcome Pack, 4–12%, 10-well 1 kit XP0412A

Novex™ WedgeWell™ Welcome Pack, 4–12%, 15-well 1 kit XP0412C

Protein detection technical handbook now availableDownload the 84-page Protein detection technical handbook, which provides practical information on improving

protein detection and sensitivity in western blot experiments. The publication covers everything from hands-free

blot processing systems, CCD imaging, and data analysis software to protocols, troubleshooting tips, and product

selection. Download this free handbook today at thermofisher.com/detecthandbook.

Increased sample volume capacityTraditionally, researchers working with low-abundance proteins

or dilute protein samples had to compromise on target detection

limits because of small sample volume capacities on protein gels.

The Novex WedgeWell precast protein gels offer one of the high-

est sample loading capacities available in a mini-sized (8 x 8 cm),

1.0 mm thick Tris-glycine gel due to their proprietary wedge-shaped

well design (Figure 2). This novel design nearly doubles the sample

capacity without the need to increase gel thickness, thus allowing

A B1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

WedgeWell Tris-glycine gel Supplier B gel

http://thermofisher.com/novexwedgebp73

http://thermofisher.com/detecthandbook


BIOPrOBEs 73 CELL VIABILITY AND PrOLIFErATION

Plate-based viability assays are a fundamental tool in drug discovery for

evaluating the potency of compounds and the sensitivity of cell lines to

specific agents. These homogeneous assays typically measure a single

parameter—such as ATP concentration, cell membrane permeability,

or reductive capacity (redox potential)—to determine viability. Viability,

however, is not easily defined in terms of a single physiological or mor-

phological parameter, and relying on a single measure can generate

bias in the experiment.

The luciferin/luciferase bioluminescence assay provides an extremely

sensitive measure of ATP and is commonly used to determine viable

cell numbers, cell proliferation, and cytotoxicity in both bacterial and

mammalian cells. Recent reports caution, however, that results from

firefly luciferase (FLuc)–based ATP assays can be misinterpreted due

to errors introduced both by a drug’s mechanism of action and by cell

line–specific responses [1–3]. One study found that as many as 60%

of the active compounds originally identified for their antiproliferative

activity (using FLuc reporter assays) are actually compounds that inhibit

FLuc activity itself [4]. Moreover, at least 12% of the compounds in the

NIH Molecular Libraries Small Molecule Repository reportedly exhibit

FLuc-inhibiting activity [5].

Multiplex or orthogonal assay approaches, which use a com-

bination of different measures of cell health, provide more stringent

viability determinations than possible with any single-parameter assay.

Here we describe a robust and convenient multiplex viability assay

that employs both the Invitrogen™ CyQUANT™ Direct Cell Proliferation

Assay, which measures cellular DNA content and membrane integrity,

and the Invitrogen™ PrestoBlue™ Cell Viability Reagent, which detects

cell metabolism. The combination of a DNA content–based assay, which

has been shown to be among the most sensitive indicators of cell health,

and a metabolism-based assay produces an effective orthogonal assay

that provides a more complete measure of cell viability. Additionally,

because neither of these assays requires cell lysis, they can be further

multiplexed with other fluorescence-based cell function probes.

Determining cell proliferation based on DNA contentThe CyQUANT Direct Cell Proliferation Assay provides a fluores-

cence-based method for determining the number of viable cells in a

population across a range of growth conditions and cell types. This

assay uses two reagents: a green-fluorescent nucleic acid stain and a

Multiplex assays for robust cell health analysesCyQUANT Direct and PrestoBlue viability assays work together.

Figure 1. Measurements of cytotoxicity differences across different cell types

using the CyQUANT Direct assay. Jurkat cells, HEPG2 cells, human aortic

smooth muscle cells (HASMCs), and human pulmonary aortic smooth muscle cells

(HPASMCs) were seeded in 384-well plates at a density of 5,000 cells per well

with 30 µL medium containing 10% FBS. Following incubation at 37°C for 48 hr

with increasing concentrations of tamoxifen, 30 µL of CyQUANT™ Direct reagent

(a component of the CyQUANT Direct Cell Proliferation Assay Kit, Cat. No. C35011)

was added to each well. Fluorescence was measured after 60 min. Fluorescence

intensities were normalized to DMSO-alone treatment, and dose-response curves

were generated. Each point graphed represents the average of the fluorescence of

eight wells. As shown, the two primary cell types (HASMCs and HPASMCs) were

significantly more sensitive to tamoxifen than the two transformed cell lines (adherent

HepG2 and suspension Jurkat cells).

log [Tamoxifen] (μM)0.50 1.75 2.001.501.250.75 1.00 2.25

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100

80

60

40

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HPASMC

HASMC

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background suppression dye. The nucleic acid stain is a cell-permeant

dye that binds to DNA and concentrates in nuclei of mammalian

cells; binding of this dye to DNA is proportional to the amount of DNA

present in the cell and independent of the cell’s metabolic state. The

background suppression dye is impermeant to live cells; however, the

outer membranes of dead and dying cells are typically compromised,

allowing the suppression dye to enter the cells and mask the fluorescent

signal from the DNA-binding dye.

The combination of these two components produces an assay that

measures both DNA content and membrane integrity, both important

for accurate determination of viability. Because cellular DNA content is

highly regulated, the CyQUANT Direct assay can be used at multiple

time points to calculate the average proliferation rate of a cell population.

In addition, cell number determined with the CyQUANT Direct assay

can be used as a highly sensitive indicator of cytotoxicity (Figure 1).

Determining cell proliferation based on metabolismThe PrestoBlue Cell Viability Reagent is both a fluorescence- and

absorption-based reagent that is used to measure the reductive

capacity of cells. This proven cell viability indicator uses the


CELL VIABILITY AND PrOLIFErATION BIOPrOBEs 73

natural reducing power of live cells to convert resazurin to the fluo-

rescent molecule resorufin. The active ingredient in the PrestoBlue

reagent (resazurin) is a nontoxic, cell-permeant compound that is blue

in color and virtually nonfluorescent. Upon entering cells, resazurin is

reduced to resorufin, which produces bright red fluorescence. Viable

cells continuously convert resazurin to resorufin, thereby generating a

quantitative measure of viability and cytotoxicity. Damaged (which are

likely to be nonproliferating cells) and nonviable cells have decreased

reductive capacity and thus generate proportionally lower signals.

The PrestoBlue reagent provides data in as little as 10 minutes,

whereas most other commercially available resazurin-based cell viability

reagents require a 1- to 4-hour incubation. Moreover, the PrestoBlue

reagent provides a measure of relative viable cell number that can be

used to assess the average proliferation rate of the populations when

performed at multiple time points. The PrestoBlue assay exhibits linear

responses over three orders of magnitude in culture medium (Figure 2)

and is compatible with downstream functional analyses because it does

not require cell lysis.

Figure 2. Linear detection range for the PrestoBlue viability assay. PrestoBlue™

Cell Viability Reagent (Cat. No. A13261) exhibits a linear response over three orders

of magnitude, with a one-step protocol and a 10 min incubation in culture medium.

Figure 3. Multiplexing the PrestoBlue Cell Viability Assay and the CyQUANT Direct Cell Proliferation Assay. To perform the multiplex viability assay, add PrestoBlue™

reagent to drug-treated cells, incubate 10 min, and read fluorescence. Following the PrestoBlue readout, add CyQUANT™ Direct reagent to cells, incubate an additional

60 min, and re-read fluorescence. The use of the PrestoBlue and CyQUANT Direct assays together produces a viability assay that is sensitive to cell metabolism, DNA

content, and changes in membrane permeability.

Cells per well (x 103)

0.1 61.50.4 25 100

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104

105

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Add PrestoBlue reagent,incubate 10 min

Measure �uorescence (Ex/Em = 569/586 nm)

from bottom

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Add CyQUANT Direct reagent, incubate 60 min

Live cells�uoresce

red

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green

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from bottom

Multiplexing PrestoBlue and CyQUANT Direct viability assaysWe have found that the PrestoBlue and CyQUANT Direct assays

can be applied to cells in tandem (first the PrestoBlue assay and

then the CyQUANT Direct assay), producing a viability assay that

detects changes in cell metabolism, DNA content, and membrane

integrity, all in the same sample. This multiplex assay, with its simple,

high-throughput mix-and-read protocol and fluorescence readout, is

compatible with standard fluorescence-based microplate readers; no

special equipment is required. The tandem PrestoBlue and CyQUANT

Direct protocol requires no wash steps and no cell lysis. Simply add

the PrestoBlue reagent to drug-treated cells, incubate for 10 minutes,

and read the fluorescence. Following the PrestoBlue readout, add

the CyQUANT Direct reagent, incubate an additional 60 minutes, and

re-read the fluorescence (Figure 3). This combined assay can be

used on the same cell sample to generate two fluorescent signals

(a PrestoBlue signal and a CyQUANT Direct signal), both of which

produce measurements of pharmacological parameters very similar

to those obtained with the Promega CellTiter-Glo™ Luminescent Cell

Viability Assay, a homogeneous luciferase-based assay for measuring

ATP levels (Figure 4).

Assessing cytotoxicity with orthogonal assaysAn ongoing collaboration between scientists at Thermo Fisher Scientific

and the NIH/NCATS High Content Imaging & Discovery Lab aims to

explore the potential advantages of using orthogonal viability assays for

toxicity profiling. A collection of 1,408 small-molecule compounds with

known toxic mechanisms of action were tested on U2OS cells. Three

high-throughput screening assays for cytotoxicity were then performed

on the cells using the multiplexed PrestoBlue and CyQUANT Direct

viability assays, as well as the CellTiter-Glo Luminescent Cell Viability



Figure 5. Venn diagram representing the dose-response screens of over

1,000 compounds from a focused library of drugs relevant for clinical use in

cancer. U2OS cells were seeded at a density of 1,000 cells/well in 5 µL of DMEM

containing 10% fetal calf serum, grown at 37°C for 8 hr, treated with a compound,

and incubated for an additional 2 days prior to being assessed for cytotoxicity.

A collection of 1,408 small-molecule compounds with known toxic mechanisms

of action were tested in a 11-point dose response at final concentrations ranging

from 780 pM to 46 µM. Controls were used to normalize for 100% toxicity (50 µM

camptothecin) and 0% toxicity (DMSO only). High-throughput screening assays were

then performed using the combined PrestoBlue™ cell viability assay and CyQUANT™

Direct cell proliferation assay (see Figure 3), or using the Promega CellTiter-Glo™

Luminescent Cell Viability Assay. For each assay, whole-well fluorescence intensity

values were analyzed, dose-response curves were fit using in-house software [6],

the curve class was assigned and characterized [2,7], and the three-way Venn

diagram was computed. Data used with permission from Steve Titus and Rajarshi

Guha, NIH/NCATS High Content Imaging & Discovery Lab, Rockville, Maryland.

References1. Thorne N, Shen M, Lea WA et al. (2012) Chem Biol 19:1060–1072.

2. Chan GK, Kleinheinz TL, Peterson D et al. (2013) PLoS One 8:e63583.

3. Leitão JM, Esteves da Silva JC (2010) J Photochem Photobiol B 101:1–8.

4. Thorne N, Auld DS, Inglese J (2010) Curr Opin Chem Biol 14:315–324.

5. Auld DS, Southall NT, Jadhav A et al. (2008) J Med Chem 51:2372–2386.

6. Southall NT, Jadhav A, Huang R et al. (2009) Chapter 19. Enabling the large-scale analysis of quantitative high-throughput screening data. Handbook of Drug Screening, Second Edition, Seethala R, Zhang L, Eds, CRC Press, 442–464.

7. Inglese J, Auld DS, Jadhav A et al. (2006) Proc Natl Acad Sci U S A 103:11473–11478.


CyQUANT™ Direct Cell Proliferation Assay 10 microplates100 microplates

C35011C35012

PrestoBlue™ Cell Viability Reagent 25 mL100 mL

A13261A13262

395

823

51 188

59

94

CellTiter-Glo

CyQUANT Direct

PrestoBlue

Assay. Of the 1,408 compounds tested, 590 displayed no cytotoxic

effect in any of the assays. Of the remaining 818 active compounds,

only 395 (48%) were identified by all three tests (Figure 5). Work is

in progress to further characterize the toxic compounds identified as

active in only one or two of the cytotoxicity assays. The goal of these

Drug concentration (µM)

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10–210–4 100 102 104

50

100

150

0

Doxorubicin (0.68 µM) Sanguarine chloride (0.6 µM)Niclosamide (1.3 µM)

Drug concentration (µM)N

orm

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esp

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10–2 100 102 104

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Drug concentration (µM)

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A. PrestoBlue assay B. CyQUANT Direct assay C. CellTiter-Glo assay

Figure 4. Pharmacological testing with three different cell viability assays. HeLa cells were exposed to three different drugs at the indicated concentrations for 24 hr.

The cells were then assayed using either the combined (A) PrestoBlue™ cell viability assay and (B) CyQUANT™ Direct cell proliferation assay (see Figure 3), or (C) the

CellTiter-Glo™ cell viability assay. The combined assay can generate two fluorescent signals (a PrestoBlue signal and then a CyQUANT Direct signal), both of which are very

similar to the results produced by the CellTiter-Glo assay. IC50 values for each drug are shown in parentheses in the key for each assay.

additional analyses is to understand their mechanisms of action and

also to explain false hits (both positive and negative) and singleton

compounds (i.e., those identified as active in only a single assay).

Cell viability assays for every applicationMultiplex viability assays can provide measures of different aspects

of cell viability, producing a more informative picture of the state of

cells. Find out more about our wide selection of cell viability assays,

available for microplate, imaging, and flow cytometry platforms, at

thermofisher.com/cellviabilitybp73. ■

Acknowledgments: Crit ical input and guidance were kindly provided by

Steve Titus and Rajarshi Guha, High Content Imaging & Discovery Lab,

National Center for Advancing Translat ional Sciences (NCATS), 9800

Medical Center Drive, Rockvi l le, Maryland.

http://thermofisher.com/cellviabilitybp73



Proliferating and apoptotic cells revealedClick-iT EdU and Click-iT TUNEL colorimetric assays for immunohistochemistry.

Labeling of cells and tissues with several dis-

tinct fluorescent probes is now a standard lab

practice that allows for multiplexing of func-

tional and antibody-based markers. However,

there are fields of research and types of

analyses that are much more amenable to

colorimetric labeling. Colorimetric detec-

tion is far more widespread when studying

highly autofluorescent tissue, such as heart,

because this autofluorescence can signifi-

cantly interfere with a fluorescent probe signal.

Colorimetric detection is also favored when

labeling must be compatible with standard his-

tological stains that provide morphological and

contextual information. Here we describe the

conversion of our popular fluorescence-based

click chemistry assays for cell proliferation and

apoptosis to colorimetric assays.

Adapting the click reaction for colorimetric detectionThe most direct assay for cell proliferation is

the measurement of new DNA synthesis via

incorporation of a thymidine analog into the

growing DNA strand during the S phase of the

cell cycle. In the click chemistry–based cell

proliferation assay, the thymidine analog EdU

(5-ethynyl-2´-deoxyuridine) is incorporated into

newly formed DNA [1]. After cells are fixed

and permeabilized, the incorporated EdU is

detected by a brief click reaction with a small

fluorescent azide, resulting in the covalent

attachment of the fluorophore to the DNA. To

convert this simple and elegant reaction to a

colorimetric signal requires the attachment of

an enzyme such as horseradish peroxidase

(HRP) to the incorporated EdU, which can

then enzymatically convert a substrate such

as diaminobenzidine (DAB) to a colored product that is deposited locally and visualized with

a white-light microscope.

Unique advantages of colorimetric click reactionsWith the development of colorimetric click assays, the click chemistry technology is now

accessible to labs that have a brightfield microscope; no specialized fluorescence equip-

ment or training is required. In addition, colorimetric signals create a permanent record on

the microscope slide that is not susceptible to fading or photobleaching, allowing results to

be viewed immediately after staining or at some later date. Furthermore, EdU-labeled cells

detected with a colorimetric DAB reaction can be subsequently treated with standard histology

stains—for example, hematoxylin and eosin (H&E)—to reveal the context and location of DNA

replication with brightfield illumination.

Colorimetric options for both Click-iT EdU proliferation assays and Click-iT TUNEL apoptosis assaysDetection of new DNA synthesis with the Invitrogen™ Click-iT™ EdU Colorimetric IHC Detection

Kit is accomplished with two rapid and essentially irreversible reactions. The click reaction

between the incorporated EdU and biotin azide forms a covalent triazole ring, which can then

bind a streptavidin-HRP conjugate, resulting in the attachment of HRP to the newly synthesized

DNA. Incubation of the cells or tissues with a solution of DAB, a widely used chromogenic HRP

substrate, results in deposition of the dark brown polymer in the nuclei of proliferating cells

(Figure 1A). Moreover, we have shown that the signals from our colorimetric Click-iT EdU assay

Figure 1. Correlation between colorimetric and fluorescence-based detection with Click-iT EdU prolifer-

ation assays. An 8 µm formalin-fixed, paraffin-embedded (FFPE) section of rat intestine was pulsed with EdU for

2 hr using the Click-iT™ EdU Colorimetric IHC Detection Kit (Cat. No. C10644). (A) The tissue was labeled with

biotin azide, streptavidin-HRP, and DAB (according to the Click-iT EdU colorimetric protocol) to reveal dark brown

nuclei, counterstained with hematoxylin (blue), dehydrated, and hard-mounted in Thermo Scientific™ Cytoseal™

60 Mounting Medium. The brightfield image was acquired using a 20x objective on the EVOS™ FL Auto Imaging

System equipped with a color camera. (B) Prior to the colorimetric detection of the EdU-labeled DNA with biotin

azide, the tissue was labeled with Alexa Fluor™ 488 azide (a component of the Click-iT™ EdU Alexa Fluor™ 488

Imaging Kit, Cat. No. C10337), and the green-fluorescent nuclei were imaged on the EVOS FL Auto Imaging

System using the FITC (green) channel and the monochrome camera.



are highly correlated with those from our fluorescence-based Click-iT

EdU assay (Figure 1B). By reacting EdU-labeled tissue with a mixture

of biotin azide and an Alexa Fluor™ azide (molar ratio of approximately

2:1), we can detect proliferation on both fluorescence and brightfield

imaging platforms using the Invitrogen™ EVOS™ FL Auto two-camera

system (which has a monochrome camera for imaging fluorescent

signal and a color camera for imaging colorimetric signal) (Figure 1).

The Invitrogen™ Click-iT™ TUNEL Colorimetric IHC Detection Kit,

which detects double-stranded DNA breaks that are the hallmark of

late-stage apoptosis, uses the same strategy as the Click-iT EdU

colorimetric assay. In the case of TUNEL (terminal deoxynucleotidyl

transferase (TdT)–dUTP nick end labeling), a TdT-tailing reaction is used

to incorporate EdUTP at the 3´ ends of the double-stranded DNA breaks.

Once this ethynyl group reacts with biotin azide through a click reac-

tion, labeled DNA is detected after incubation with a streptavidin-HRP

conjugate and DAB, resulting in colorimetric detection in the nuclei of

late-stage apoptotic cells (Figure 2).

Strategies for gaining access to EdU-labeled DNAThe conventional antibody-based BrdU proliferation assay labels newly

synthesized DNA with the thymidine analog 5-bromo-2´-deoxyuridine

(BrdU) and then detects the incorporated BrdU with a labeled anti-BrdU

antibody. Antibody-based cell- and tissue-labeling protocols typically

entail some form of antigen retrieval, most commonly heat-induced

epitope retrieval (HIER) or protease digestion (PIER), to disrupt

fixative-induced crosslinking and restore epitopes so that they can

be recognized by the antibody. In addition to HIER, BrdU detection

requires that cells or tissues be additionally treated with acid, heat, or

DNase to denature the DNA and allow the anti-BrdU antibody access

to the incorporated analog.

Because it does not rely on antibodies for detection, fluores-

cence-based Click-iT EdU detection does not require any extraordinary

antigen retrieval or DNA denaturation treatments; for formalin-fixed,

paraffin-embedded (FFPE) tissue, deparaffinization and incubation

with the small fluorescent azide are all that are needed before imag-

ing the incorporated EdU. For colorimetric Click-iT EdU detection,

the pretreatment requirements are intermediate between those for

antibody-based BrdU assays and fluorescence-based EdU assays.

With colorimetric Click-iT assays, no acid or enzyme treatment is

required for DNA denaturation but either HIER or PIER must be applied

for the streptavidin-HRP conjugate to gain access to the site of the

click-modified DNA. However, the type and duration of this antigen

retrieval treatment is very permissive. Although trypsin is supplied in

the kit, alternative methods of HIER and PIER have been evaluated and

shown to be sufficient to result in detectable EdU signals. For labs that

have already determined the HIER methods useful for their antibodies

of interest, such protocols will likely also work for colorimetric Click-iT

EdU detection. Alternative PIER protocols work equally well—both

trypsin or proteinase K digestion have been tested. For delicate tissue

like embryonic mouse heart, a short 5-minute protease digestion is

sufficient; longer digestion of 20 to 30 minutes is appropriate for more

durable tissue like adult rat uterus or intestine.

Contextual information with histology stainsAs with any multicolor experiment, detection of multiple targets requires

contrasting and non-overlapping colors; when colocalizing two signals,

the lighter color is usually assigned to the counterstained background

while the darker color is designated for the signal of interest so that it

masks the background. The commonly used H&E counterstain works

well with DAB-based Click-iT colorimetric assays. Methyl green coun-

terstain also contrasts well with the DAB signal and is compatible with

Click-iT EdU and Click-iT TUNEL colorimetric assays, but it requires

nonaqueous mounting conditions.

Figure 2. Mouse tissue section labeling with the colorimetric Click-iT TUNEL

apoptosis assay. An 8 µm formalin-fixed, paraffin-embedded (FFPE) section of

mouse thymus was labeled with DAB using the Click-iT™ TUNEL Colorimetric IHC

Detection Kit (Cat. No. C10625) to reveal apoptotic cells (dark brown nuclei). The

tissue was subsequently stained with eosin Y (pink) followed by nuclear counterstain-

ing with methyl green (blue), dehydrated, and hard-mounted in Thermo Scientific™

Cytoseal™ 60 Mounting Medium. The brightfield image was acquired using a 20x

objective on the EVOS™ FL Auto Imaging System equipped with a color camera.



More complex histological stains are

useful when identifying key components

such as collagen, elastin, muscle, and mucin

found in some tissues. The DAB signal pro-

duced by the Click-iT EdU colorimetric assay

is compatible with Movat’s pentachrome

stain—a five-color stain for nuclei and elastin

(black), collagen (yellow), ground substance

and mucin (blue), fibrin (bright red), and

muscle (red)—with only a slight adaptation

of the staining protocol. One component of

Movat’s pentachrome stain—Verhoeff’s elastin

stain—results in black nuclei as well as black

elastin. By over-differentiating with the ferric

chloride mordant (i.e., washing out the extra

black staining of the nuclei by slightly extend-

ing the ferric chloride rinse), the nuclei can be

lightened to pale gray while the elastin (with

its higher affinity for the stain) remains black.

This lightening of the nuclear staining allows

the dark brown DAB proliferation signal to be

seen within the light gray nuclei. Thus, tissues

can be click-labeled with EdU or EdUTP for

the Click-iT proliferation or apoptosis assay,

respectively, and then the labeled DNA is

detected using the HRP substrate DAB to

Figure 3. Use of colorimetric Click-iT EdU labeling combined with Movat’s pentachrome stain to show details of proliferation in different tissue types. (A) A 7 µm

formalin-fixed, paraffin-embedded (FFPE) section of mouse embryonic heart valve (age PNDO) labeled with DAB using the Click-iT™ EdU Colorimetric IHC Detection Kit

(Cat. No. C10644) and counterstained with Movat’s pentachrome stain reveals the nuclei of proliferating cells containing EdU-labeled DNA (dark brown) together with elastin

(black) and collagen (yellow) along the edge of the valve. Also visible are red blood cells (bright red), muscle (dark red), and nuclei of nonproliferating cells (gray). (B) The

same staining strategy was applied to an FFPE section of rat intestine. DAB labeling identifies the nuclei of proliferating cells (dark brown), and the pentachrome stain reveals

goblet cells (blue), collagen (yellow), muscle (dark purple-red), red blood cells (bright red), and the nuclei of nonproliferating cells (gray-purple).

Figure 4. Peyer’s patch in mouse intestine showing both apoptotic and proliferating cells. (A) An 8 µm

formalin-fixed, paraffin-embedded (FFPE) section of mouse intestine containing a Peyer’s patch was treated with

citrate HIER and then labeled with rabbit primary antibody that recognizes cleaved caspase-3, followed by Alexa

Fluor™ 594 goat anti–rabbit IgG secondary antibody (Cat. No. A11012, pink). The tissue was counterstained with

Hoechst™ 33342 (Cat. No. H1399, blue). Autofluorescence in the FITC channel (green) shows general tissue

morphology and background fluorescence and helps to eliminate false-positive caspase signals. The tissue sec-

tion was mounted in ProLong™ Gold Antifade Mountant (Cat. No. P10144) and imaged on the EVOS™ FL Auto

Imaging System using a 20x objective. (B, C) Mouse intestinal tissue containing a Peyer’s patch was labeled with

DAB using either (B) the Click-iT™ TUNEL Colorimetric IHC Detection Kit (Cat. No. C10625), indicating the DNA

fragmentation in the nuclei (dark brown) of apoptotic cells, or (C) the Click-iT™ EdU Colorimetric IHC Detection Kit

(Cat. No. C10644), indicating newly synthesized DNA in the nuclei (dark brown) of proliferating cells. In each case,

tissues were dehydrated, and hard-mounted in Thermo Scientific™ Cytoseal™ 60 Mounting Medium. The brightfield

images were acquired using 20x objective on the EVOS FL Auto Imaging System equipped with a color camera.

produce a dark brown signal, followed by modified Movat’s pentachrome staining, which

produces colors that contrast well with the DAB staining. Using this staining strategy, we can

detect proliferating cells within the heart valve, in the context of elastin, collagen, and the

surrounding muscle tissue (Figure 3A), as well as within intestinal tissue (Figure 3B).

Peyer’s patches exhibit both proliferation and apoptosisClick chemistry does double duty within special areas of the small intestine containing lymph

regions known as Peyer’s patches that function in immune surveillance of and response to

the contents of the intestinal lumen. Within the Peyer’s patches, both proliferating cells and




Click-iT™ EdU Colorimetric IHC Detection Kit 1 kit C10644

Click-iT™ TUNEL Colorimetric IHC Detection Kit 1 kit C10625

References1. Salic A, Mitchison TJ (2008) Proc Natl Acad Sci U S A 105:2415–2420.

2. Van der Loos CM, Becker AE, Van den Oord JJ (1993) Histochem J 25:1–13.

Figure 5. Two-color dual-pulse labeling of proliferating cells in rat tissue using

EdU and BrdU. After pulsing with EdU, followed by BrdU 3 days later, rat tissue was

pretreated according to a standard BrdU protocol, which requires trypsin digestion

followed by HCl denaturation. The Click-iT EdU labeling reaction was performed

prior to overnight incubation with an anti-BrdU primary antibody (clone MoBU-1,

Cat. No. B35128), which does not cross-react with EdU. Secondary labeling steps

containing streptavidin-HRP and alkaline phosphatase (AP) goat anti–mouse IgG

antibody were combined. EdU was detected with Vector™ NovaRED™ Peroxidase

Substrate (red-brown), an alternative HRP substrate, using the staining protocol for

the Click-iT™ EdU Colorimetric IHC Detection Kit (Cat. No. C10644), followed by

BrdU detection using Vector™ Blue Alkaline Phosphate Substrate (blue). EdU-labeled

cells (pulsed first, red-brown) are distributed throughout the vaginal epithelial layer,

while BrdU-labeled cells (pulsed 3 days later, blue) appear adjacent to the basement

membrane. This stained tissue section was wet-mounted in PBS and imaged using

a 20x objective on the EVOS™ FL Auto Imaging System.

apoptotic cells are normally present. Fluorescence-based antibody

staining that detects cleaved caspase-3 indicates that there are cells

undergoing the early to middle stages of apoptosis, when the caspase

family of enzymes is activated (Figure 4A). Colorimetric Click-iT

TUNEL labeling also shows the presence of late-stage apoptotic

cells that contain fragmented DNA (Figure 4B). Proliferating cells

can be detected with colorimetric Click-iT EdU labeling (Figure 4C).

When using colorimetric staining for both TUNEL and EdU assays on

the same tissue, we recommend first click-labeling the proliferating

cells in the Peyer’s patches with EdU and biotin azide and detecting

the EdU-labeled cells using streptavidin-HRP and DAB. This EdU

assay can then be followed by a second click-labeling reaction of

the apoptotic cells by TdT-tailing of the fragmented DNA with EdUTP,

followed by reaction with biotin azide and detection with streptavi-

din-HRP and an HRP substrate that produces a red or purple stain

(such as Vector™ NovaRED™ or Vector™ VIP HRP substrates, from

Vector Laboratories).

Two-color pulse labeling of DNA with EdU and BrdUThe colorimetric Click-iT EdU assay and the antibody-based BrdU assay

can be used together in a dual-pulse labeling experiment, as long as

chromatically distinct enzymatic products will be detected. To detect

proliferation in tissue that has been pulsed first with EdU and then with

BrdU, we used the antigen retrieval and DNA denaturation treatments

required for BrdU detection, followed by reaction of EdU with biotin azide

and incubation of BrdU with the anti-BrdU primary antibody (typically

overnight at 4°C). Next, the tissue was incubated with a mixture of a

streptavidin-HRP conjugate (which binds the biotin) and an alkaline

phosphatase (AP) conjugate of a secondary antibody (which binds the

anti-BrdU primary antibody), followed by their respective substrates,

to produce two-color dual-pulse labeling of proliferation (Figure 5).

The HRP and AP substrates were chosen carefully to ensure that their

products would be chromatically distinct and chemically compatible;

note that the AP reaction must be performed in the absence of phos-

phate buffer. Only a few color combinations permit the determination

of colocalization from both chromogens without the use of spectral

deconvolution methods [2].

In an alternative approach, both the EdU and BrdU thymidine ana-

logs can be labeled with HRP, but this labeling must be done sequentially,

with an enzyme-quenching step (often with hydrogen peroxide) between

successive staining steps. This single-enzyme approach offers a broader

choice of colored HRP substrates and better solvent compatibility, but

it requires more steps in the detection protocol.

Learn more about click chemistry–based assaysWe have a wide selection of Click-iT kits for detecting proliferation

and apoptosis on multiple platforms. Learn more about the new colo-

rimetric detection kits and all of the click chemistry–based assays at

thermofisher.com/clickitbp73. ■

Acknowledgments: Crit ical input and guidance were kindly provided

by the Stankunas and Powell labs at the Institute of Molecular Biology,

University of Oregon, Eugene, Oregon.

http://thermofisher.com/clickitbp73



Accurately measure cell concentrations by flow cytometryHigh-speed cell counting with the Attune NxT Flow Cytometer.

The Invitrogen™ Attune™ NxT Flow Cytometer is a next-generation cytometer that uses

traditional hydrodynamic focusing for low sample rates (10–20 μL/min, Figure 1A) and

acoustic-assisted focusing for high sample rates (100–1,000 μL/min, Figure 1B). Acoustic-

assisted hydrodynamic focusing is a revolutionary technology that precisely align cells using

ultrasonic radiation pressure (>2 MHz) to transport particles into the center of the sample

stream. This prefocused stream is then injected into the sheath stream, which supplies

additional hydrodynamic pressure to the sample prior to interrogation with one or more lasers

(Figure 1B). The combination of these two forces results in a narrow particle stream and

uniform laser illumination, regardless of the sample input rate.

In cytometers that rely solely on hydrodynamic focusing, the sample is spread across a

wider core stream as the flow rate increases, which results in less uniform laser illumination

and broader population distributions (Figure 1B). To obtain optimal data with the lowest sig-

nal variability, a conventional flow cytometer must be run at the lowest sample rate, typically

10–20 μL/min; higher sample rates produce greater variability and less precise measurements.

With acoustic-assisted hydrodynamic focusing, the Attune NxT cytometer avoids this compro-

mise between data quality and sample rates by uncoupling cell alignment from sheath flow.

Another important advantage of the Attune NxT cytometer over traditional flow cytometers

is its unique volumetric sample and sheath fluid delivery system. Samples are introduced to

the Attune NxT cytometer with syringes, producing accurate measurements of the volumes

of acquired samples, which in turn leads to accurate calculation of cell concentrations

(conveniently displayed as events/μL by the Attune NxT software). In contrast, conventional

hydrodynamic focusing systems do not measure the volume of the acquired sample. Instead,

counting beads must be added to calibrate the acquired volumes before calculating cell

concentrations, which can lead to the introduction of errors from pipetting, gating, and extra

calculations. Here we present two experiments where the Attune NxT cytometer’s increased

sampling rate and precise sample volume measurements produce highly accurate cell con-

centration data in dilute samples.

Measure the concentration of lymphocyte subpopulationsHuman lymphocyte subpopulations were iden-

tified and their concentrations measured using

a 6-color antibody panel for the common lym-

phocyte phenotypic markers CD3, CD4, CD8,

CD19, CD45, and CD56 to identify helper

T cells, suppressor T cells, B cells, and natural

killer (NK) cells. The high sampling rate of the

Attune NxT cytometer precluded the need to

wash the whole blood during the protocol,

minimizing cell loss. The choice of excitation

channels provided by the Attune NxT cytometer

equipped with 4 lasers (405, 488, 561, and

637 nm) permitted the selection of fluorescent

CD-specific antibodies that did not display sig-

nificant spectral overlap, eliminating color com-

pensation and simplifying the workflow. Figure 2

shows the scatter plots and cell concentrations

for all lymphocyte subpopulations. The accuracy

and precision of the cell concentrations obtained

in this experiment were verified by running the

sample at three different flow rates (100, 200,

and 500 µL/min) (Figure 3).

Analyze bacteria in wastewaterA sample of municipal wastewater was labeled

with the Invitrogen™ LIVE/DEAD™ BacLight™

Bacterial Viability Kit to identify live and dead

bacteria. On traditional flow cytometers, very

dilute samples can take a long time to acquire

due to slower flow rates. This sample (3 mL)

was analyzed on the Attune NxT cytometer at

a flow rate of 1 mL/min, which allowed quick

analysis of the sample and accurate detection

of very small quantities of bacteria. Moreover,

we determined the concentrations of the live

and dead bacteria without using reference

counting beads.Figure 1. Traditional hydrodynamic focusing vs. acoustic-assisted hydrodynamic focusing.

B. High sample rates (100–1,000 µL/min) A. Low sample rates (10–20 µL/min)

Traditional hydrodynamic focusing

Acoustic-assisted hydrodynamic focusing

Traditional hydrodynamic focusing

Acoustic-assisted hydrodynamic focusing

Acoustic�eld

Hydrodynamic core

Laser(cross-section)

Laser(cross-section)

Acoustic�eld

Hydrodynamic core

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Figure 4 shows the two-parameter dot plot (propidium iodide vs. SYTO™ 9 fluorescence), with

the live (green) and dead (red) bacterial populations well separated; the statistics table displays

the concentration measurements for the labeled bacteria. Wastewater may also include small

eukaryotes and types of bacteria that are potentially viable but nonculturable, each of which

may also be labeled with the dyes; the grey dots represent debris found in the wastewater.

Obtain high sample rates and accurate cell concentrationsWith its high sampling rates and ability to accurately measure sample volume, the Attune NxT

Flow Cytometer not only permits more streamlined workflows but also improves the accuracy

of cell concentration measurements. Find out more about the Attune NxT Flow Cytometer and

its software capabilities at thermofisher.com/attunebp73. ■

Figure 2. Lymphocyte subset analysis on the Attune NxT Flow Cytometer. A 100 µL aliquot of normal

human whole blood was labeled with fluorophore-conjugated antibodies against CD surface markers, followed

by red blood cell lysis using 2 mL of High-Yield Lyse Fixative-Free Lysing Solution (Cat. No. HYL250), resulting

in a 1:21 dilution of the blood. The sample was acquired on the 4-laser Attune™ NxT Flow Cytometer (Cat. No.

A24858). (A) Lymphocytes are identified on a density plot of CD45 vs. side scatter with an oval gate around

the lymphocyte (CD45+) population. (B) Cells in the lymphocyte gate are displayed on a density plot of CD3

vs. side scatter. Rectangle gates surround the CD3+ T cell and CD3– B and natural killer (NK) cell populations.

(C) Cells in the CD3+ gate are then displayed on a density plot of CD4 vs. CD8 to quantify CD4+ helper T cells

(CD4+ CD3+ CD45+) and CD8+ cytotoxic T cells (CD8+ CD3+ CD45+). (D) CD3– cells are displayed on a density

plot of CD56 vs. CD19 to distinguish CD56+ NK cells from CD19+ B cells. The statistics table shows the gat-

ing and measured concentrations (cells per µL). The product list at the end of this article shows the antibody

conjugates used.


Attune™ NxT Flow Cytometer—Blue/Red/Violet/Yellow Lasers 1 each A24858

LIVE/DEAD™ BacLight™ Bacterial Viability Kit, for microscopy and quantitative analysis 1 kit L7012

Mouse Anti–Human CD3 delta Antibody (Clone 7D6), APC conjugate 0.5 mL MHCD0305

Mouse Anti–Human CD4 Antibody (Clone S3.5), Alexa Fluor™ 488 conjugate 0.5 mL MHCD0420

Mouse Anti–Human CD8 Antibody (Clone 3B5), PE-Cy®7 conjugate 0.5 mL MHCD0812

Mouse Anti–Human CD19 Antibody (Clone SJ25-C1), Pacific Blue™ conjugate 0.5 mL MHCD1928

Mouse Anti–Human CD45 Antibody (Clone HI30), Pacific Orange™ conjugate 0.5 mL MHCD4530

Figure 3. Replicate samples collected at three

flow rates on the Attune NxT Flow Cytometer.

In the experiment described in Figure 2, cell con-

centrations were measured using three different

flow rates: 100, 200, and 500 µL/min. The Attune™

NxT Flow Cytometer provides similar concentration

measurements for each lymphocyte subpopulation,

regardless of the flow rate. Each bar represents the

mean cells/µL ± standard deviation of three samples

run at each indicated flow rate for each population.

Figure 4. Analysis of bacteria in treated municipal

wastewater on the Attune NxT Flow Cytometer.

A sample of treated municipal wastewater was

labeled with a 1:1 mixture of the cell-permeant,

green-fluorescent SYTO™ 9 and cell-impermeant,

red-fluorescent propidium iodide (PI) stains (compo-

nents of the LIVE/DEAD™ BacLight™ Bacterial Viability

Kit, Cat. No. L7012), incubated for 15 min protected

from light, and then acquired on the Attune™ NxT Flow

Cytometer at a flow rate of 1 mL/min with a forward

scatter threshold. The blue 488 nm laser was used

with a 530/30 nm bandpass emission filter for SYTO 9

detection in BL1, and a 695/40 nm emission filter

for PI detection in BL3. SYTO 9–stained live (green)

and PI-stained dead (red) populations of bacteria are

demonstrated in a dual-parameter dot plot of SYTO 9

vs. PI fluorescence. Grey dots represent debris pres-

ent in the wastewater sample. The statistics table

shows the concentration of live (1 cell/µL) and dead

(3 cells/µL) bacteria in the acquired sample.

CD56+CD8+CD4+ CD19+

5

0

10

15

20

25

Con

cent

ratio

n (c

ells

/µL)

100 µL/min

200 µL/min

500 µL/min

SYTO 9 �uorescence

Pro

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104 105 106

102

103

104

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CD56 PE �uorescence (YL1)

CD

19 P

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)

0

104

105

106

0 104103 105 106–103

CD19: 8.9

CD56: 17.2

CD4 Alexa Fluor 488 �uorescence (BL1)

CD

8 P

E-C

y7

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(YL4

)

0

0

105

105

104

104

106

106

CD8: 16.9

CD4: 42.0

CD3 APC �uorescence (RL1)

Sid

e sc

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r (x

103 )

0

1,000

500

0 105104 106

CD3–: 29.6 CD3+: 62.2

CD45 Paci�c Orange �uorescence (VL1)

Sid

e sc

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r (x

103 )

0102 103 104 105

1,000

500 Lymphocytes91.9

A B C

D

http://thermofisher.com/attunebp73



Figure 2. Quantitative analysis of protection from photobleaching by ProLong

Live Antifade Reagent for key dyes and fluorescent proteins. HeLa or U2OS

cells were stained with (A) Hoechst™ 33342 (Cat. No. H21492), (B) CellLight™

Mitochondria-RFP (Cat. No. C10505), (C) CellLight™ Mitochondria-GFP (Cat. No.

C10508), or (D) MitoTracker™ Green FM (Cat. No. M7514) reagents. Cells were

incubated for 2 hr in the dark either in complete medium (control) or in complete

medium containing ProLong™ Live Antifade Reagent (Cat. No. P36974). After incu-

bation, cells were imaged every 15 sec with the Thermo Scientific™ ArrayScan™ VTI

HCS Reader, using optimal but consistent excitation/emission imaging conditions.

0

0.5

1

1 21 41 61 81 101 121 141 161 181

120% more images

Number of images

D MitoTracker Green FM

ProLong LiveControl

0

0.5

1

1 21 41 61 81 101 121 141 161 181 201 221

1 21 41 61 81 101 121 141 161 181 201 221

50% moreimages

CellLight Mitochondria-GFP

Number of images

Nor

mal

ized

�u

ores

cenc

e in

tens

ity

C

ProLong LiveControl

CellLight Mitochondria-RFP

0

0.5

1

1 21 41 61 81 101 121 141 161

100% more images

B

ProLong LiveControl

0

0.25

0.5

0.75

1

190% more images

Hoechst 33342A

Nor

mal

ized

�u

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e in

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ity ProLong LiveControl


ProLong™ Live Antifade Reagent, for live-cell imaging 1 mL 5 x 1 mL

P36975 P36974

Protection from photobleaching for live-cell imagingProLong Live Antifade Reagent is here.One of the key pain points when labeling cells and tissues with fluo-

rescence is photobleaching, the degradation of fluorescent signals

as samples are exposed to light. Photobleaching is a complex photo-

dynamic process whereby a photoexcited fluorophore interacts with

molecular oxygen, resulting in the destruction of the fluorophore and

the production of highly reactive singlet oxygen (1O2) that can further

degrade neighboring dye molecules. This loss of signal is particularly

problematic when attempting to collect time-course data, image

rare targets with low signal-to-noise ratios, or quantitatively compare

fluo rescently labeled samples. The recently introduced Invitrogen™

ProLong™ Live Antifade Reagent takes a novel approach to protect fluo-

rescent proteins and dyes from photobleaching in live cells (Figure 1).

How ProLong Live Reagent protects live cellsWhile researchers using fixed-cell systems have long had the luxury of

a number of commercial antifade mounting media to choose from—

such as Invitrogen™ ProLong™ Diamond Antifade Mountant—these

formulations are not compatible with live-cell systems. Moreover, the

protection provided by homemade antifade formulations—such as

Trolox™ antioxidant [1,2] or ascorbic acid—is not significant for most

fluorescent dyes and virtually nonexistent for fluorescent proteins.

Based on Oxyrase™ antioxidant technology [3], ProLong Live Antifade

Reagent contains enzymes isolated from E. coli plasma membranes that

metabolize environmental components that exacerbate photobleaching.

Furthermore, these enzymes are not cell permeant, so intracellular func-

tions are minimally affected. ProLong Live Antifade Reagent is simply

diluted into cell medium or a suitable imaging buffer, and then added

directly to cells for a 15- to 120-minute incubation. After incubation,

imaging can be performed for up to 24 hours with continuous protection

from photobleaching. ProLong Live Antifade Reagent has been validated

to provide protection for a wide range of organic dyes as well as fluo-

rescent proteins (Figure 2). Best of all, ProLong Live Antifade Reagent

has been rigorously tested and shows little to no measurable effect on

cell vitality, proliferation, or incidence of apoptosis for at least 48 hours.

Protect your live cells from photobleachingProLong Live Antifade Reagent can help you maintain longer imaging

times for scans or time-lapse experiments, and enable you to detect

low-abundance targets without sacrificing cell health. To learn more,

go to thermofisher.com/prolonglivebp73. ■

Figure 1. Multicolor live-cell imaging with ProLong Live Antifade Reagent.

U2OS cells were transduced with CellLight™ Talin-GFP (Cat. No. C10611, green) and

CellLight™ Golgi-RFP (Cat. No. C10593, orange) for 24 hr, labeled with MitoTracker™

Deep Red FM (Cat. No. M22426, purple) and NucBlue™ Live ReadyProbes™ Reagent

(Hoechst™ 33342, Cat. No. R37605, blue), and incubated with ProLong™ Live Antifade

Reagent (Cat. No. P36974) for 90 min before imaging on a confocal microscope.

References1. Rasnik I, McKinney SA, Ha T (2006) Nat Methods 3:891–893.

2. Cordes T, Vogelsang J, Tinnefeld P (2009) J Am Chem Soc 131:5018–5019.

3. Thurston M, Maida D, Gannon C (2000) Lab Med 31:509–512.

http://thermofisher.com/prolonglivebp73



First generation

Secondgeneration

Thirdgeneration

FourthgenerationA

Brightness

Num

ber

of c

ells

B

Tracking cell division with flow cytometryA guide to studying cell proliferation by generational tracing.Cell proliferation assays provide a critical piece of the puzzle when evaluating cell

health, cytotoxicity, and the efficacy of anti-cancer drugs. Moreover, much of our

current knowledge of the immune system derives from the ability to initiate an ex

vivo immune response in isolated T lymphocytes, triggering their proliferation. By

labeling cells in a population with a fluorescent dye that is divided evenly between

two daughter cells following cell division, researchers can quantify cell proliferation

through multiple generations using a high-throughput fluorescence-based platform

such as flow cytometry (Figure 1).

CFSE: The original cell tracerSince its first reference as a cell tracing reagent in 1994 [1], carboxyfluorescein diac-

etate succinimidyl ester (CFDA SE, or CFSE) has been cited thousands of times in the

literature for both in vivo and in vitro cell proliferation studies. CFSE is a cell-permeant,

nonfluorescent dye that enters the cell by diffusion through the plasma membrane.

Once inside the cell, this nonfluorescent molecule is converted to a fluorescent deriv-

ative by intracellular esterases. At the same time, the active succinimidyl ester group

covalently binds to accessible protein amines, resulting in long-term retention of the

fluorescent conjugate within the cell. Through subsequent cell divisions, daughter cells

receive approximately half of the fluorescent label of their parent cells. Flow cytometric

analysis of the fluorescence intensities of individual cells in a population permits the

determination of the number of generations through which a cell has progressed since

the label was applied. Despite its increased popularity over time, CFSE has a number

of technical limitations that should be considered when choosing a fluorescent tracer

for cell proliferation measurements.

CellTrace dyes: Improved cell tracersLike CFSE, the dyes in the Invitrogen™ CellTrace™ Cell Proliferation Kits—including

CellTrace Violet, CellTrace Yellow, and CellTrace Far Red—facilitate both in vivo and in

vitro cell proliferation analyses by flow cytometry. CellTrace dyes easily cross the plasma

membrane and covalently bind inside cells, where the stable, well-retained fluorescent

conjugate offers a reliable fluorescent signal without significantly affecting morphology or

physiology. Importantly, these dyes produce fluorescent staining with very little variation

between cells within a generation, allowing each generation to be reliably distinguished.

The intense fluorescent staining provided by CellTrace dyes permits the visualization

of proliferating cells through a minimum of six generations, even after several days in a

cell culture environment or following fixation. There are several criteria to consider when

choosing the right cell tracer for an experiment, and the following sections focus on the

most important technical factors. Also see page 30 for a summary of a recent journal

article that discusses methods to assess cell tracing reagents.

Cell tracers must not be toxic to live cellsCell tracers are used on living, dividing cells and

therefore must not have a significant impact on

cell health and viability. For example, the concen-

tration of the traditional cell tracer CFSE must be

carefully optimized for each cell line and culture

condition because high concentrations of CFSE

have been reported to impact cell viability and

division, whereas low concentrations may limit

the number of cell generations detected [1]. At

the recommended working concentration of 5 µM,

CFSE-labeled Jurkat cells had less than 15%

viability after 6 days of staining (Figure 2). Titration

studies of CFSE labeling of Jurkat cells demon-

strated that concentrations between 0.5 µM and

1 µM were optimal for maintaining cell viability,

and concentrations above 2 µM induced

Figure 1. Proliferation analysis using a fluorescent

cell tracer that partitions evenly between daughter

cells. (A) Illustration of dye dilution over progressive cell

divisions. (B) Flow cytometric analysis reveals a bright,

homogeneous fluorescent signal from the initial population

of cells. Subsequent cell divisions result in larger numbers

of cells possessing half the fluorescence intensity of their

parent cells.



BA

Control 5 µM 5 µM 10 µM 1 µM

CellTrace CFSE

CellTrace Violet

CellTrace Yellow

CellTrace Far Red

% D

ead

cel

ls

Cell tracer concentration

20

0

40

60

80

100

Control 5 µM 2 µM 1 µM 500 nM

CellTrace CFSE

20

0

40

60

80

100


% D

ead

cel

ls

Figure 2. Viability analysis of Jurkat cells labeled with fluorescent cell tracers. (A) Jurkat cells were labeled

with CellTrace™ CFSE (Cat. No. C34554) at concentrations between 5 µM and 0.5 µM for 20 min, diluted 5-fold

with fresh culture medium (containing protein to bind any free dye), and incubated for 6 days before viability

analysis using SYTOX™ Red Dead Cell Stain (Cat. No. S34859); control cells were incubated with 5 µM DMSO.

(B) Jurkat cells were labeled with the recommended concentrations of CellTrace CFSE, CellTrace™ Violet (Cat.

No. C34557), CellTrace™ Yellow (Cat. No. C34567), or CellTrace™ Far Red (Cat. No. C34564) dye, and then

incubated for 6 days before viability analysis.

Figure 3. Monitoring the initial decrease in fluorescence for three cell tracers. (A) U937 cells were left

unlabeled or labeled with CellTrace™ CFSE (Cat. No. C34554), CellTrace™ Violet (CTV, Cat. No. C34557), or

CellTrace™ Far Red (CTFR, Cat. No. C34564) dye at a concentration of 10 µM and then analyzed on a BD™ LSR II

flow cytometer on the indicated day after treatment. (B) Histogram showing representative fluorescence mea-

surements for U937 cells labeled with CTFR. In this histogram, data from each successive day in the experiment

are shown as follows: red = day 0, orange = day 1, green = day 2, blue = day 3, violet = day 4; for reference,

the fluorescence distribution from unlabeled U937 is also presented on the overlay histogram (grey). Data used

with permission from Joseph D. Tario Jr. and Paul K. Wallace, Roswell Park Cancer Institute, Buffalo, New York.

minimal transfer between adjacent cells [2].

Consequently, CellTrace Violet dye provides

better resolution between division peaks

and therefore more accurate quantitation in

generational analysis.

Cell tracers must exhibit bright and stable fluorescence in labeled cellsTo enable detection of cel l prol i feration

through many generations, the ideal cell

tracer should produce a bright fluorescent

signal that is stil l detectable after several

rounds of division, despite the dilution of

the signal with each successive generation.

CFSE-labeled cells, however, show a signif-

icant decrease in fluorescence after the first

24 hours. It has been reported that this is

likely due to the cells’ clearing of short-lived

proteins that were initially labeled with the

dye [3]. Although this loss of fluorescence

may not affect every experiment in which

CFSE is used, it suggests that there will

be less tracer available for generational

analysis over time. In contrast, indepen-

dent test ing by Joseph Tario Jr. at the

Roswell Park Cancer Institute demonstrates

that, unlike CFSE, neither CellTrace Violet

dye nor CellTrace Far Red dye shows the

unwanted initial decrease in fluorescence

(Figure 3).

Multiplexable cell tracers increase options in panel designCell proliferation assays are often used in

concert with other cell function assays to

provide a more informative picture of the

state of the cell. Although widely used for

proliferation analysis, CFSE (excitation/

emission = 492/517 nm) requires one of

the most popular flow cytometry channels—

the same channel used by FITC, Alexa Fluor™

488 dye, and GFP. In contrast, the CellTrace

Day 0

Day 1

Day 4

Unstained 10 µMCFSE

10 µMCTV

10 µM CTFR

Fluo

resc

ence


101

100

102

103

104

Cel

l num

ber

00 101 102 103 104–101

200

400

600

800

1,000

CellTrace Far Red �uorescence

A B

toxicity (Figure 2A). In contrast, the other CellTrace dyes did not exhibit toxic side effects

after 6 days of staining at the concentrations tested (Figure 2B). In particular, CellTrace

Violet, CellTrace Yellow, and CellTrace Far Red showed minimal effects on cell viability at

concentrations of 5 µM, 10 µM, and 1 µM, respectively, substantially increasing the window

of effective reagent concentration for cell proliferation analysis.

Cell tracers must show minimal cell-to-cell dye transferCFSE is known to undergo nonspecific cell-to-cell dye transfer. This dye transfer decreases

the observed resolution of cell generations, thereby reducing accuracy when identifying cell

division populations. Although most succinimidyl esters also undergo membrane-exchange

mechanisms that negatively affect peak resolution, CellTrace Violet dye is unique in that it shows



Figure 4. Multiplex flow cytometry analysis of cell proliferation. Human peripheral blood mononuclear cells isolated from whole blood were stained with 10 µM CellTrace™

Yellow dye (provided in the CellTrace Yellow Cell Proliferation Kit, Cat. No. 34567) and then resuspended in fresh medium. Stained cells were stimulated to proliferate using

Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Cat. No. 11161D) and incubated at 37°C and 5% CO2 for 6 days. After incubation, cells

were fed 10 µM EdU (provided in the Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit, Cat. No. C10632) for 2 hr, washed, stained with LIVE/DEAD™ Fixable

Near-IR Dead Cell Stain (Cat. No. L34975) for 30 min, washed, and fixed for 15 min in 4% formaldehyde. Cells were treated with the Click-iT reaction cocktail containing

Alexa Fluor 488 azide for 30 min to complete the click reaction, labeled with APC mouse anti–human CD4 antibody for 20 min, and washed. Finally, cells were labeled with

FxCycle™ Violet Stain (Cat. No. F10347) for 30 min and analyzed on a 4-laser Attune™ NxT Flow Cytometer (with 405, 488, 561, and 638 nm lasers, Cat. No. A24858).

(A) This dual-parameter density plot shows newly synthesized DNA, as determined with the Click-iT Plus EdU assay, vs. DNA content, as determined with FxCycle Violet

Stain. It is used to analyze the cell cycle phases of proliferating cells in the population. (B) This dual-parameter density plot shows DNA content, as determined with FxCycle

Violet Stain, vs. progression of generations, as determined with CellTrace Yellow dye. This plot displays the progression of cell generations on the x-axis and DNA content on

the y-axis. Cells that are not currently dividing (G0/G1) are shown in blue; cells from each generation that are actively synthesizing DNA or are about to divide are seen at the

top of the plot. (C) This dual-parameter density plot shows the S-phase analysis provided by Click-iT EdU labeling vs. the generational analysis provided by CellTrace Yellow

labeling. The parent-cell generation can be seen on the far right of the plot, with successive generations represented by clusters of dots to the left of the initial generation.

Cells from each generation that are actively synthesizing DNA are seen at the top of the figure.

Product Laser type Ex/Em* Quantity Cat. No.

CellTrace™ Violet Cell Proliferation Kit, for flow cytometry 405 nm 405/450 180 assays20 assays

C34557C34571

CellTrace™ Yellow Cell Proliferation Kit, for flow cytometry 532 or 561 nm 546/579 180 assays C34567

CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry 488 nm 492/517 180 assays20 assays

C34554C34570

CellTrace™ Far Red Cell Proliferation Kit, for flow cytometry 633 or 635 nm 630/661 180 assays20 assays

C34564C34572

*Fluorescence excitation (Ex) and emission (Em) maxima, in nm.

References1. Lyons AB, Blake SJ, Doherty KV (2013) Curr Protoc Cytom Chapter 9:9.11.1–9.11.12.

2. Filby A, Begum J, Jalal M et al. (2015) Methods 82:29–37.

3. Wallace PK, Muirhead KA (2007) Immunol Invest 36:527–561.

4. Quah BJ, Parish CR (2012) J Immunol Methods 379:1–14.

CellTrace Yellow �uorescence

Clic

k-iT

Plu

s E

dU

A

lexa

Flu

or 4

88 �

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scen

ce

102 103 104 105

101

102

103

104

CellTrace Yellow �uorescence

FxC

ycle

Vio

let

�uor

esce

nce

104

105

102 103 104 105

FxCycle Violet �uorescence (x 103)

Clic

k-iT

Plu

s E

dU

A

lexa

Flu

or 4

88 �

uore

scen

ce

5 15 25 3510 20 30 40

101

102

103

104

A B C

Violet, CellTrace Yellow, and CellTrace Far

Red dyes are excited by the 405 nm, 532

(or 561) nm, and 633 (or 635) nm lasers,

respectively. Not only do these tracers allow

for greater flexibility in flow cytometry panel

design, but each dye can be multiplexed with

488 nm–excitable green-fluorescent probes.

Figure 4 shows a multiplex experiment that

incorporates CellTrace Yellow dye for mea-

suring cell proliferation, Invitrogen™ FxCycle™

Violet Stain for fixed-cell DNA content analysis,

the Invitrogen™ Click-iT™ Plus EdU Alexa

Fluor™ 488 Flow Cytometry Assay Kit for

detecting newly synthesized DNA, Invitrogen™

LIVE/DEAD™ Fixable Near-IR Dead Cell Stain

for assessing viability, and a fluorescent anti-

body conjugate for immunophenotyping. This

multicolor analysis demonstrates the depth

of information that can be gathered when

additional functional reagents are used to

assay the cell population of interest.

Explore the CellTrace Cell Proliferation KitsWhen compared with traditional cell tracers such as CFSE, the CellTrace dyes provide signif-

icant advantages, including their low toxicity and bright and stable fluorescence. In particular,

CellTrace Violet dye has been cited in several comparative studies as superior to CFSE for cell

proliferation analyses [2,4]. Learn more about the entire line of CellTrace Cell Proliferation Kits

at thermofisher.com/celltracebp73. ■

Acknowledgments: Crit ical input and guidance were kindly provided by Joseph D. Tario Jr. and

Paul K. Wallace, Roswell Park Cancer Institute, Buffalo, New York.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Immunological+Investigations%2C+36%3A527%E2%80%93561%2C+2007%2C+title%3A+Cell+Tracking+2007%3A+A+Proliferation+of+Probes+and+Applications%2C+authors%3A+Paul+K.+Wallace+and+Katharine+A.+Muirhead

http://www.ncbi.nlm.nih.gov/pubmed/?term=Quah+BJ%2C+Parish+CR%2C+J+Immunol+Methods+2012%2C+379%2C+1-14

http://thermofisher.com/celltracebp73


JOUrNAL CLUB BIOPrOBEs 73

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilutionFilby A, Begum J, Jalal M, Day W (2015) Methods 82:29–37.

While regulated cell proliferation provides the

foundation for normal growth and develop-

ment, cell cycle dysregulation is a hallmark of

cancer and autoimmune diseases. The use of

high-throughput, fluorescence-based meth-

ods such as flow cytometry is paramount to

the study of cell proliferation. Key to prolifera-

tion analysis is the ability to uniformly label live

cells with a fluorescent dye and then clearly

identify distinct generations of cells through

time by following the decrease in fluorescent

signal as it is diluted in daughter cells. Without

adequate resolution of the division peaks

(Figure 1), it is difficult to assess quantitative

cell proliferation data accurately using dye

dilution, even with the use of fitting algorithms.

The cell tracing dye carboxyfluorescein

diacetate succinimidyl ester (CFDA SE, or

CFSE) is among the earliest and most popular

reagents used in cell cycle studies [1]. With

the development of CFSE alternatives that do

not require 488 nm excitation, cell proliferation

assays are now more amenable to multiplex

analyses with other fluorescent probes. Filby

and coworkers recently published a method to

assess and compare the performance of five

commercially available fluorescent cell tracing

reagents: three succinimidyl ester–based dyes

(Invitrogen™ CellTrace™ Violet and CellTrace™

Far Red dyes and Affymetrix™ Cell Proliferation

Dye eFluor™ 670) and two lipophilic dyes

(CellVue™ Claret and PKH26 reagents), none

of which requires 488 nm excitation. Jurkat

cells were used in this study because these

transformed heterogeneous, nonquiescent

cells provide a more challenging model system

than relatively uniform primary T and B cells

and, when labeled, invariably exhibit a broad

fluorescence distribution. For each of the dyes,

performance testing included:

■ Labeling efficiency, via measurement of

viability, intensity, and uniformity of the

dye-labeled cells

■ Need for cell sorting to resolve division

peaks

■ Dye-spec i f i c con t r ibu t ion to any

re-spreading error of the sorted populations

■ Nonspecific cell-to-cell dye transfer

■ Symmetrical inheritance across the cyto-

kinetic plane

The authors observed that , a l though

culture-dependent sources of error exist, suc-

cinimidyl ester–based dyes were superior for

cell proliferation studies when compared with

lipophilic dyes. Lipophilic dyes required higher

labeling concentrations to achieve comparable


CellTrace™ Violet Proliferation Kit, for flow cytometry

180 assays20 assays

C34557C34571

Reference1. Quah BJC, Parish CR (2012) J Immunol Methods

379:1–14.

Figure 1. Tracking cell division in lymphocytes

using CellTrace Violet dye. Human peripheral

blood lymphocytes were harvested and stained

with CellTrace™ Violet dye (Cat. No. C34557). The

violet peaks represent successive generations of

cells stimulated with anti–human CD3 antibody and

interleukin-2 (IL-2) and cultured for 7 days. The peak

outlined in black represents cells that were cultured

for 7 days with no stimulus. Data were collected on a

BD™ LSR II Cell Analyzer and analyzed using Attune™

cytometer software. Reproduced from The Molecular

Probes Handbook: A Guide to Fluorescent Probes and

Labeling Technologies, 11th edition (2010).

CellTrace Violet �uorescence

Num

ber

of c

ells

1000

14

27

41

54

101 102 103 105104

staining intensity and uniformity, and they

produced more culture-dependent variation

that impacted peak resolution. However,

except for CellTrace Violet dye, the succin-

imidyl ester–based dyes also demonstrated

various suboptimal attributes. CellTrace Violet

is the only dye that did not transfer between

cells in culture; moreover, CellTrace Violet

distributed across the cytokinetic plane in

a highly symmetrical fashion, resulting in

well-resolved division peaks. The authors

state, “Collectively, these data support our

current view that of the dyes we have tested,

[CellTrace Violet dye] still remains the best

for tracking proliferation in any amenable

cell type by fluorescent dye dilution and flow

cytometry.” This conclusion is in line with an

earlier study by Quah and coworkers [1] that

also found CellTrace Violet dye to be “the best

available alternative to CFSE in the analysis

of cell divisions.”

In conclusion, Filby and colleagues sug-

gest that the choice of labeling dye and, when

necessary, the use of cell sorting to narrow

the width of the parent cell population are

critical steps when conducting cell proliferation

experiments. These steps should ensure that

division peaks can be well resolved and that

any algorithms applied to the data will generate

accurate cell parameters, including percentage

of divided cells and proliferation index. ■


BIOPrOBEs 73 JOUrNAL CLUB

Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatmentsDe Biasi S, Cerri S, Bianchini E et al. BMC Med (2015) 13:277.

Idiopathic pulmonary fibrosis (IPF) is a rare but

devastating lung disease in which the pulmo-

nary vasculature and other tissues undergo a

profound remodeling that produces a severe

decline in breathing eff iciency. IPF usual ly

occurs in adults with a history of cigarette

smoking, an activity that damages lung tis-

sue. Individuals with IPF have f ibrotic lung

regions with very few blood vessels, adjacent

to unaffected tissue that is highly vascularized.

Evidence suggests that when re-endothelization

fails to occur after alveolar injury, the resulting

loss of alveolar–capillary integrity may trigger

the process of fibrosis.

It is suspected that circulating endothelial

cells (CECs), endothelial progenitor cells (EPCs),

and fibrocytes may be correlated with response to

vascular injury and to tissue repair that occurs in

the lungs, even though each of these populations

makes up less than 1% of circulating nucleated

cells in blood. If so, endothelial cells may poten-

tially be used as biomarkers of IPF disease pro-

gression and prognosis. However, data on CECs,

EPCs, and fibrocytes from individuals with IPF

have been very difficult to acquire. These cells

are exceedingly rare, so their detection requires

the sensitive analysis of a large number of cells.

De Biasi and coworkers recently published

methods to measure CEC and EPC popula-

tions, as well as fibrocytes, from individuals with

IPF. To identify the rare cells in freshly isolated

blood samples, they designed multiparametric

labeling schemes for the target populations and

took advantage of the speed and precision of

the Invitrogen™ Attune™ NxT Flow Cytometer

(Figure 1). The Attune NxT cytometer permitted

the researchers to acquire phenotypic data on

35,000 cells per second, which enabled them


Attune™ NxT Flow Cytometer—Blue/Red/Violet/Yellow Lasers 1 each A24858

LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation

80 assays200 assays400 assays

L34973L10120L34974

Figure 1. Gating strategy for the identification of circulating endothelial cells (CECs) and endothelial

progenitor cells (EPCs). Debris, monocytes, and dead cells were excluded by use of an electronic gate

and dump channel, containing cells identified by an anti-CD14 monoclonal antibody and a viability marker

(LIVE/DEAD™ Fixable Far Red Dead Cell Stain, Cat. No. L10120). CECs and EPCs were identified on the

basis of expression of CD45, CD34, and CD133: CECs were defined as CD45dim, CD34+, and CD133−,

while EPCs were defined as CD45−, CD34+, and CD133+. The expression of CD309 (VEGFR-2, KDR) was

detected among EPCs and CECs. For this phenotype analysis, cells were acquired using a 14-color, 4-laser,

high-speed Attune™ NxT Flow Cytometer (Cat. No. A24858). Reprinted with permission from De Biasi S, Cerri

S, Bianchini E et al. (2015) BMC Med 13:277, and under the Creative Commons Attribution 4.0 International

License (http://creativecommons.org/licenses/by/4.0/).

CD309-FITC �uorescence

Sid

e sc

atte

r

CD309-FITC �uorescence

Sid

e sc

atte

r

Sid

e sc

atte

r

CD133-APC �uorescenceCD133-APC �uorescence

Sid

e sc

atte

r

CD45-PE �uorescence

CD

34-P

C7

�uor

esce

nce

Dump(CD14-APCVio770,

LIVE/DEAD Fixable Far Red Dead Cell Stain)

Sid

e sc

atte

r

Time

Sid

e sc

atte

r

Forward scatter

Sid

e sc

atte

r

CECEPC

to analyze the more than 10 million cells per sample needed for a meaningful statistical

analysis of such small numbers of target events. Additional research will be needed to

determine whether characteristics of these rare circulating cells can be used as biomarkers

of IPF progression and prognosis, but De Biasi and coworkers have demonstrated that such

research is possible with tools available today. ■


IN MEMOrIAM BIOPrOBEs 73

“Hey you, get to work”; that’s my endearing memory of Mike. As we built

CellLight™ reagents, I took a trip to his lab—the mecca of imaging—

and never regretted it. Four days of tissue culture, microscopy, and

beer & BBQ, and I knew we had a viable product. He was the coach

that you always wanted to play for, but you also knew it was going to

take everything you had to make an impression. At the end of those

four days he said don’t hesitate to come back or send more product for

testing. I was as proud of that work as anything I’ve done in my career

in science. His journey from his Georgia roots to world-class scientist

was inspirational to us all, and I miss his counsel already.

—Mike O’Grady, Senior Manager, Research & Development

Mike’s work has significantly contributed to making imaging an integral

part of life science research while always pushing the boundaries of its

applications. At the same time, he helped bring the wonders of natural

science to a lay audience, and his colorful ties with micrograph images

are but one example of that. Mike was an approachable expert whose

research and collaborations across academia and industry helped

inform, educate, and enable scientists worldwide. His long-standing

collaboration with Molecular Probes benefited not only our scientists

but also our customers.

—Magnus Persmark, Senior Product Manager, Protein and Cell Analysis

A tribute to our colleague Mike Davidson

L ate last year the fluorescence microscopy community lost one of its most

celebrated, experienced, and passionate members. On December 24,

2015, Michael W. Davidson passed away in Tallahassee, Florida. During an

extremely productive career, Mike Davidson authored well over 100 research papers

in peer-reviewed journals, and his scientific guidance was even highlighted at the

2014 Nobel Prize awards by Eric Betzig, who shared the Nobel Prize for Chemistry.

Mike’s impressive number of publications is really just the tip of the iceberg; it doesn’t

tell the whole story of his enormous contribution to the field. He created educational

information for Nikon, Olympus, and Zeiss as well as for his own website—Molecular

Expressions™ (micro.magnet.fsu.edu)—through Florida State University, providing

an invaluable resource for fluorescence microscopists, beginners and experienced

scientists alike. Mike was also a long-time commercial partner to us at Thermo

Fisher Scientific, manufacturing our FluoCells™ slides and providing images for the

Molecular Probes Handbook (including our latest cover) and BioProbes Journal.

Mike, however, was much more than that; he was a trusted advisor, patient teacher, collaborator, and friend, providing feedback without agenda

and always in an open and honest manner. That is how many of us at the legacy Molecular Probes site in Eugene, Oregon will remember him.

—Nick Dolman, Senior Staff Scientist, Protein and Cell Analysis

What I remember most about Mike is the time I visited him at his labs

in Florida. There was an array of new, high-tech microscopy hardware

and software donated by imaging companies that were hoping to have

Mike and his team use them to push the boundaries of imaging and

get some of those coveted high-resolution, eye-popping microscopy

images of cells and tissues. There was information about the latest

fluorescent proteins and their uses. There was Mike’s intense and

high-energy work ethic. And there were dress ties and other materials

decorated with images of polarized crystals of popular alcoholic bev-

erages and compounds, which he sold (and often gave away). I still

have the one he gave to me.

—Jason Kilgore, Technical Applications Scientist, Technical Support

Mike was that special breed of scientist that combined deep scientific

expertise with pragmatism for results. The impact Mike had on the fluo-

rescence microscopy community and the use of fluorescent proteins

as tools for cell biology is second only to the mentoring and inspiration

he gave to many scientists through his collaborative spirit. Amidst all

of the scientific discussions, what I remember most about Mike is his

passion for teaching and his sensibilities in problem solving; whatever

the project or question, he was a master at paving a path forward.

—Mike Janes, Senior Manager, Research & Development

http://micro.magnet.fsu.edu

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