FlowCytometry 2013-Handout

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2

......the same applies to Cell Biology

Cell Surface Marker:

Fluorochrome labeledantibodies

enable the characterizationof thecellular PHENOTYPE either byFlow

Cytometry or Imaging (e.g. CD4:CD8 Ration, HIV Patients)

Membrane:

Membrane analysis ist oftenused in the fieldof:

Ion Chanel and Transport

Structural integrity

Apoptosis and Necrosis

Live/Dead

Flow Cytometry or Imaging application

Nucleus:

DNA-specific and -reactive Dyes can be used

for DNA content analysis for:

Cell Cycle Analysis

Live/Dead Detection

Proliferation

and can be detected either

by Flow Cytometry or Imaging

IL-12

INF-y

IL-4

Intra Cellular Analysis:

Fluorochrome labeledantibodies enable the

characterization of Intracellular Structresand/or Proteins e.g. Cytokines and can be

detected either by Flow Cytometry or

Imaging (e.g. IL-12, INF-y uponst activation)

Cellular Analysis Flow Cytometry Applications

DNA (cell cycle analysis, proliferation)

Intracellular staining (cytokines, antigens)

Functionality tests

Proliferation

Apoptosis

Redox Potential

pH

Phagocytosis

Ion Indicators (e.g. Calcium)

Surface staining (Phenotyping)

Cell Membrane (Structural Integrity)

Flow Cytometry , Clinical Aspekts

Stem Cells (CD34)

DNA content (ploidity)

Multi Drug Resistance (MDR)

Cytokine Detection (IC, EC)

Subtype Classification (e.g. Treg, DC)

Crossmatching

Phenotyping (e.g.Transplantation [HLA], HIV)

CancerFlow Cytometry

� Cell Analysis

� Subclass Isolation

� Single Cell Suspension

� Fluorescence Based

� Fluid Stream

� Single Cell Measurement

� Size of the Cell

� Granularity of the Cell

� Fluorescence

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Sheath Fluid

Light SourceDetection

Laminar

Flow

Laminar

Flow

Sheath Fluid

SAMPLE

PMTPhoto

Multiplier

Tube

A/DDatatranslation

Data

Analysis

The Flow Cytometry Principle

Analyzed Parameters:

Forward Scatter (FSC)

Size of the cell

Side Scatter (SSC)

Granularity of the cells

Fluorescence

Intensity

Number of Events

Fl-1, Fl-2, Fl-3, Fl-4 etc.

LASER LIGHT (e.g. 488nm)

The Flow Cytometry Principle

The The Scatter Scatter Principle (FSC, SSC)Principle (FSC, SSC)

Right Angle Light Detector

Side Scatter (SSC)

Cell surface, Granularity

Forward Light Detector


Cell size

Light Source


Sid

e S

catt

er

(SS

C)

Granulocytes

Monocytes

Lymphocytes

The Forward/Side Scatter Principle (FSC, SSC)

Dot Blot

Sample; Lysed Whole Blood

granularitycell surface

Relative Size

What is a Dot BlotWhat is a Dot Blot• Depicts individual events (particles or cells) versus two detected parameters

• A standard Dot Blot used in Flow Cytometry is the so called Scatter Blot,consisting of the Forward Scatter (FSC) Information and the Side Scatter(SSC)

• Therefore each “Dot” on a Scatter Blot stands for a measured event(typically Cells) and its SSC value and FSC value.

• Another typically used parameter in a Dot Blot is Fluorescence Intensity

• In a standard assay a typical number of events measured is 100.000

Flu

ore

sce

nce

Inte

nsi

ty

Fluorescence IntensityForward Scatter (FSC)

Sid

e S

catt

er

(SSC

)

What is a Quadrant BlotWhat is a Quadrant Blot

• A Quadrant Blot (QB) is a Dot Blot, segmented into four sections

• The QB is an ideal tool to analyze ( as %) individual events versus twoparameters

• Typically this tool is used to analyze populations versus two fluorochromes(targets)

LL

UR

LR

UL

UL = upper left

UR = upper right

LL = lower left

LR = lower right

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Data Acquisition, HistogramData Acquisition, Histogram

Time

Vo

lta

ge

T ime

Vo

lta

ge

T ime

Vo

lta

ge

Sample FlowVoltage Pulse in PMT

Peak:

Height

Width

Area

Laminar

Flow

Sheath Fluid

SAMPLE

What is a Histogram PlotWhat is a Histogram Plot

• The Histogram is a graphical representation showing a visual impression of the distribution of data, here Fluorescence Intensity

• The Fluorescence Intensity is proportional to the number of Binding sites

• The Peak symmetry (height/width=area) can also be used to analyse

• The reference here is the unstained cell (ideally using an Isotype Control)

15

The Flow Cytometry Workflow

Sample Collection

Sample Analysis

Cell Viability

Sample Preparation Extracellular Staining

Intra Cellular Staining

Block Unspecific Binding

Cytokines/Growthfacors/Hormons

DNA fragmentation

Cell Functionality

FOR INTERNAL USE ONLY


Fc-Blocking Reagents

• Block non-specific Fc-mediated binding of antibodies

• Reduce background and improve resolution of flow cytometry data

Inhibit the non-specific Fc-gamma receptor (FcγR)

� optimal staining of selective antibody

� optimal signal to noise ration (decrease background)

eBioscience Blocking Reagents:

Human FcγR-Binding Inhibitor

anti Mouse CD16/32-Block Fc-Binding

Intra-/Extra-cellular Staining

Lysis of Red Blood Cells

� Reduced Backround

� Optimized Signal to Noise

� Optimized Scatter Plot

Intra-/Extra-cellular Staining

Intracellular Staining Buffers

� Maintain Scatter Characteristics after Fix/Perm

� No effect on targeting structure

� Cytosolic Proteins

� Cytokines

� Nuclear Factors

� Transcription Factors

� Optimized access to target structure (Antibody access)

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� Simultaneous analysis of surface molecules and intracellular antigens

� Single cell level

� 1st Surface Stain

� 2nd Fixation (to stabilise structure)

Formaldehyd mediated

� 3rd Permeabilisation (to allow access for mAb)

Saponin mediated

� Stimulation required due to low initial levels of target protein by resting cells

� PMA (phorbol ester, protein kinase activator)

� Ionomycin (calcium ionophore)

� anti-CD3

� Lipopolysaccharide (LPS)

� T-cell stimulation to produce INF-γ, TNF-α, IL-2 and IL-4

� PMA, Ionomycin

� anti CD3

� Monocyte stimulation to produce IL-6, IL-10 or TNF-α

� LPS

� Transport Inhibitors (block secretion)

� Monensin

� Brefeldin A

The Flow Cytometry Workflow

Sample Collection

Sample Analysis

Cell Viability

Sample Preparation Extracellular Staining

Intra Cellular Staining


Cytokines/Growthfacors/Hormons

DNA fragmentation

Cell Functionality

The eBioscience Knowledge Center

www.eBioscience.com


www.eBioscience.com


www.eBioscience.com


www.eBioscience.com

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www.eBioscience.com

Detection of Detection of Antigens (EC/IC)Antigens (EC/IC)

Specific Probe or Antibody

Dye/Fluorochrome

Introduction to Fluorescence TechniquesIntroduction to Fluorescence Techniques

Fluorescence DetectionFluorescence Detection

Four essential elements of fluorescence detection systems can be

identified from the preceding discussion:

1) Excitation source

2) Fluorophore

3) Wavelength filters

to isolate emission photons from excitation photons

4) Detector

that registers emission photons and produces a recordable output,

usually as an electrical signal or a photographic image. Regardless of

the application, compatibility of these four elements is essential for

optimizing fluorescence detection.

Fluorescence InstrumentationFluorescence Instrumentation

Fluorescence instruments are primarily of four types, each providing distinctly different information:

Spectrofluorometers and microplate readers

measure the average properties of bulk (µL to mL) samples.

Fluorescence microscopes

resolve fluorescence as a function of spatial coordinates in two or three dimensions for microscopic objects (less than ~0.1 mm

diameter).

Fluorescence scanners

including microarray readers, resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects

such as electrophoresis gels, blots and chromatograms.

Flow cytometers

measure fluorescence per cell in a flowing stream, allowing

subpopulations within a large sample to be identified and quantitated.

......it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.............it’s all about fluorescence.......

The Fluorescence Process

• Fluorescence is the result of a three-stage process

• Fluorescence occurs in rigid molecule structures

• Fuorescent molecules are in general: polyaromatic hydrocarbons or heterocycles

• These molecules are called fluorophores or fluorescent dyes.

• The fluorescence process is illustrated by the simple electronic-state diagram

_(Jablonski diagram)

Phenolphthalein

Flexible molecule, non-fluorescent

Fluorescein

Rigid molecule, fluorescent

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The Fluorescence Process: Jablonski Diagramm

Stage 1 : Excitation (external source, idealy at the Exitation maximum)

Stage 2 : Excited-State Lifetime (finite 1-10ns)

Stage 2 : collision quenching

fluorescence resonance energy transfer (FRET)

intersystem crossing

Stage 3 : Fluorescence Emission

Tandem ConjugatesTandem Conjugates

Fluorescence Resonance Energy Transfer (FRET)

R-PE, PerCp, APC Cy 5, Cy 5.5, Cy 7, Alexa Fluor 700

488nm

Excitation

Far Red

EmissionDonor

Dy e

Acceptor

Dy e

Energy Transfer

Fluorescent Dyes in Immune Diagnostic

Multicolour Analysis = Multiple Parameters

34

Fluorescence IntensityFluorescence Intensity

Nu

mb

er

of

eve

nts

Fluorescent intensity

100 102101 104103 105

Fluorescence Intensity is proportional to the number of binding sites

35

Fluorescence Intensity and BrightnessFluorescence Intensity and Brightness

Relative Brightness:

PE - APC – PerCP- PE-Cy5.5 - PE-Cy5 - PE-TR - PE-Cy7 - APC-Cy7 - FITC

Bright Dim

Nu

mb

er

of

eve

nts

Fluorescent intensity

100 102101 104103 105

Defining bright versus dim: Stain IndexDefining bright versus dim: Stain Index

Stain Index (SI) = D/W

D = difference between pos and neg peak medians

W = 2 x robust SD

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Fluorescence Intensity and BrightnessFluorescence Intensity and Brightness

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

Fluorescent DyesFluorescent Dyes

FFluoresceinluoresceinIIsosoTThiohioCCyanatyanat (FITC)(FITC)

Xanthendye

excitation: 488 nmemission max.: 518 nm

molecular weight: 0.389 kDa

Detected in FL-1 channel in most instruments

Prone to Photobleaching

Intensity sufficient for

strong expressed AG

Staining of human platelets with purified

mouse IgG1 isotype control (cat.#14-4714)

(dotted histogram) or purified HIP8 (solid

histogram) followed by FITC anti-mouse IgG

(cat.#11-4011 ). Total viable cells were used for

analysis.

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

AAlexalexa FFluorluor®® 488488 (AF 488)(AF 488)

Modified Xanthendye

excitation: 488 nmemission max.: 518 nm


Detected in FL-1 channel in most instruments

Excellent Photostability

Staining of HeLa cells with Alexa

Fluor® 488 Mouse IgG2a, K Iso Cntrl

(cat. 53-4724) (open histogram) or

Alexa Fluor® 488 anti-human

MICA/M ICB (6D4) (colored

histogram). Total viable cells were

used for analysis.

OH2

N

SO3

-SO

3

-

NH2

+

CO2

-

O

O

N

O

O

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

RedRed--PPhycohycoEErythrinrythrin (R(R--PE)PE)

Phycobiliprotein(Cyanobacteria)

Oscillatoria rubescens

excitation: 488 nm

emission max.: 575 nm

molecular weight: > 240 kDa

Very bright signal, high quantum yield

Prone to FotobleachingStaining of permeabil ized C57Bl/6

thymocytes (left) and permeabil ized Jurkat

(right) cells with 0.5 µg of PE Mouse IgG1,

K Iso Cntrl (cat. 12-4719) (open histogram)

or 0.5 µg of PE 1E7.2 (colored histogram).

Total cells were used for analysis. 1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

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PE PE PE PE –––– Texas Red (PETexas Red (PETexas Red (PETexas Red (PE----TR)TR)TR)TR)

excitation: 488 nm

emission max.: 615

molecular weight: > 240 kDa +TR

Detected in FL3 on single laser

instruments

PE overlap requires compensation

PE PE PE PE –––– AlexaAlexaAlexaAlexa Fluor 610 (PEFluor 610 (PEFluor 610 (PEFluor 610 (PE----AF610)AF610)AF610)AF610)

excitation: 488 nm

emission max.: 628

molecular weight: > 240 kDa +AF610

Alternative to PE-TR

Less overlap into PE channel due to

628nm emission

Less subject to Fc mediated binding

than PE-Cy5

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

PEPE--Cy5Cy5 (Cy(Cy--ChromeChrome®®, TRI, TRI--COLORCOLOR®®, PC5, PC5®®))

Tandem Conjugate: R-PE and Cy5

excitation: 488 nm


molecular weight: R-PE + X Cy5 > 242 kDa

Detected in FL3 or FL4 in most instruments

When used with APC in dual laser instruments,

inter laser compensation is required

Staining of normal human peripheral blood

cells with PE-Cy5 Mouse IgG1, K Iso Cntrl

(cat. 15-4714) (open histogram) or PE-Cy5

anti-human CD2 (RPA-2.10) (colored

histogram). Total cells were used for

analysis.

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

PE PE PE PE ---- Cy5.5Cy5.5Cy5.5Cy5.5

excitation: 488 nm



Detected in FL3 or FL4 in most

instruments

When used with APC in dual laser

instruments, inter laser compensation is required

Better choice for those instruments than

PE-Cy5

Pe rCPPe rCPPe rCPPe rCP ---- Cy5.5Cy5.5Cy5.5Cy5.5

excitation: 488 nm



Detected in FL3 or FL4 in most

instruments

When used with APC in dual laser

instruments, inter laser compensation is required

Better choice for those instruments than

PE-Tandems due to the larger stokes shift

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

PerCPPerCP--eFluor™710eFluor™710

Anti-mouse CD4 (clone RM4-5), anti-mouse CD8 (clone 53-6.7), and anti-human

CD3 (clone OKT3) were conjugated to either PerCP-eFluor™ 710 (red) or PerCP-

Cy5.5 (pink) for direct comparison. Mouse splenocytes were stained with anti-

CD4 (left panel) or anti-CD8 (middle panel). Human PBMCs were stained with

anti-CD3 (right panel).

Comparison of PerCP-eFluor™ 710 and PerCP-Cy5.5 Conjugates

Detected in FL3 on most instruments; tandem dye,

resonance energy transfer from PerCP molecule to

eFluor® 710; when used with APC on dual laser

machines, needs a cytometer capable of inter-laser

compensation. Better choice for dual laser

instruments than PE-tandems as a result of its large

stokes shift. 2-3 fold brighter than PerCP-Cy5.5.

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

PEPE--Cy7 Cy7 (Phycoerythrin(Phycoerythrin--Cyan7 Tandem Conjugate) Cyan7 Tandem Conjugate)

Tandem Conjugate: R-PE and Cy7

excitation: 488 488 488 488 nm nm nm nm or 530 nm530 nm530 nm530 nm



Detected in FL3 or FL4 in most instruments

Issues:

Brightness, Compensation, Stability-Fixation

eBioscience Improved PE-Cy7 Conjugations:

Brighter Staining

Less Compensation

Greater Stability

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

The histogram demonstrates staining with new

PE-Cy7 anti-human CD14 from eBioscience (red

line), or from the competitor (green line). Data

provided courtesy of Mihoko Whalen, Tobias

Kollmann and Pascal Lavoie from the Child &

Family Research Institute in Vancouver.

AAllolloPPhycohycoCCyaninyanin (APC)(APC)

Phycobiliproteinfound in blugreen algae

excitation: 633 / 635 nmemission max.: 660 nm

molecular weight: 105 kDa

Intensity sufficient for low expressed antigens

Staining of C57Bl/6 bone marrow cells with

0.125 μg of APC Rat IgG2b Isotype Control (cat.

17-4031) (blue histogram) or 0.125 μg of APC

anti-mouse Gr-1 (RB6-8C5 ) (purple histogram).

Total viable cells were used for analysis.

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

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AAlexalexa FFluorluor®® 647 (AF 647)647 (AF 647)

Carboxylic Acid, Succinimidyl Ester

excitation: 633 / 635 nmemission max.: 665 nm


AF 647 Intensity comparable to APC

Intracellular staining

Photo stability

Staining of F9 embryonal

carcinoma cells with 0.5 μg of

Alexa Fluor® 647 Rat IgG2a Iso

Cntrl (cat. 51-4321) (open

histogram) or 0.5 μg of Alexa

Fluor® 647 anti-mouse OCT3/4

(EM92) (colored histogram). Total

cells were used for analysis. 1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

eFluoreFluor®® 660 (alternative to APC or 660 (alternative to APC or AlexaAlexa FluorFluor®® 647) 647)

Human per iphera l blood monocytes w ere

stained with mous e Ig M isotype control

(dotted line) or the anti-CD36 clone NL07

conjugat ed to eFluor 660 (red histogram) or

Alexa Fluor 647 (gray histogram).

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

Immunofluorescent i ma ge of cryos ection

of mouse intestine stain ed with eFluor

660 conjugated anti-mouse L YVE-1

antibody and nuclear counterstain DAPI.

excitation: 633 / 635 nmemission max.: 660nm

Excellent Photo stability

Comparable brightness to APC

Ideal tool for IHC applications

APC-eFluor® 780(bright and stabile alternative for APC(bright and stabile alternative for APC--Cy7 or APCCy7 or APC--H7)H7)

Human PBMC

Anti CD3 (UCHT1)

APC-eFluor 780

Human PBMC

Anti CD3 (UCHT1)

APC-Alexa Fluor 750

Overlay Panel 1/2 Mouse splenocytes

Anti CD8 (53-6.7)

APC-eFluor 780 (blue)APC-H7 (red)

1 – 2 – 3 – 4 – 5 low- - - - - - - - - - - - - - - - -high

Fluorochromes for BD FACSCanto IITM

(4-2-2 Configuration)

Excitation [nm]: 488nm488-nm solid state, 20-mW

Filter: 530/30 BP

Filter: 585/42 BP

Filter: 670 LP

Filter: 780/60 BP

FITC (Fluorescein)

Alexa FluorTM 488

PE (Phycoerythrin)

Alexa FluorTM 555

PerCP-eFluor 710

PerCP

PE-Cy 7

Excitation [nm]: 633nm633-nm HeNe, 17-mW laser

Filter: 660/20 BP

Filter: 780/60 BP

APC

Alexa FluorTM 647

eFluorTM 650NC

APC-eFluor 780

APC-Cy7



Excitation [nm]: 405nm405-nm solid state diode, 30-mW

Filter: 450/50 BP

Filter: 502-525nm

eFluorTM 450

Pacific BlueTM

Fixable Viability

Dye eFluor 505



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Blue Laser OptionsBlue Laser Options RedRed Laser OptionsLaser Options

2010 | www.eBioscience.com57

Green Laser OptionsGreen Laser Options Fluorochromes example for a 7 color stain using the red

and blue laser line

How can we go further???

What about a 10 colour stain????

EMPOWER YOUR VIOLET LASER!!!

The Next Generation in FluorescenceThe Next Generation in Fluorescence

eFluoreFluor™ ™ NanocrystalsNanocrystals

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eFluoreFluorTMTM Nanocrystal CompositionNanocrystal Composition

� Core composition and size determines color of the Nanocrystal

� Inorganic shell improves stability and brightness

� Unique, propriartity lipid coating provides water solubility and functional

groups for conjugation

� Biomolecules are covalently attached to functional groups on coated

nanocrystal

Core Shell Lipid Layer

2- 10 nm

CdSe ZnS Water Soluble Nanocrystal

Semiconductor Semiconductor NanocrystalsNanocrystals; The principal ; The principal

The excited complex is a very strong reductant� photobleaching

The shell keeps the excited electron from interacting with the environment

A high-energy photon excites an

electron across the bandgap

A bandgap-energy photon is emitted as

the electron falls back to the ground stage

Semiconducting

Infinite Solid

Conduction Band

(LUMO)

Valence Band

(HOMO)

Bandgap (Eg)

Antibonding orbital

(mostly metal d character)

Bonding orbital

(mostly anion p character)

Eg(0), α and β are material constants

Semiconducting

Nanocrystal

eFluoreFluor NanocrystalNanocrystal Platform TechnologiesPlatform Technologies

Flow

Fl. MicroscopyIn Vivo

eFluoreFluor™ ™ NanocrystalsNanocrystals

• Large “Stokes Shift”

• Narrow emission = minimal compensation

• Excellent photostability

• Compatible with conventional fluorochromes/ Filter sets

Excitation/EmissionEmission Comparison

Tra

dit

ion

al

Tra

dit

ion

al

eF

luo

rS

olu

tio

n

The eFluor™ Solution: Compensation Requirements

VioletViolet Laser OptionsLaser Options

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Fluorochromes example for a 10 color stain

using the violet, red and blue laser line Conclusions:Conclusions:

• Plan your experiment, what do I want to know

• Know your cells

• Consider environmental conditions

• Sensitivity required

• Selection of dyes

• Select instrument; acquisition rate/flow rate

• Optimize staining procedure

• Titrate you antibody for optimal staining

• Data analysis

Multicolor Multicolor Flow Flow Cytometry:Cytometry:

Experimental DesignExperimental Design

Design and analysis of multicolor flow Design and analysis of multicolor flow

cytometry experimentscytometry experiments

• Design

• Instrument configuration

• Fluorochrome performance

• FluorPlan™ Spectra Viewer

• Tandem dye considerations

• Compensation considerations

• Analysis

• Eliminating false positives

• Proper controls for gating

Instrument considerationsInstrument considerations


• Lasers, PMTs, filters and dichroic mirrors

�This will define your fluorochrome choices

• Obtain baseline PMT voltage settings

�Use only as a place to start

• Use fluorescent calibration beads to understand

resolution and performance for each detector

available

Assessment of detectors on an LSR II Assessment of detectors on an LSR II

using 8using 8--peak beadspeak beadsFITC: PE: PerCP-Cy5.5: PE-Cy7:

Blue laser (488 nm)

APC: APC-eFluor 780:Red laser (633 nm)

eFluor 450: eFluor 605NC:Violet laser (405 nm)

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Fluorochrome performanceFluorochrome performance

• Understand fluorochrome performance

• Match brightest fluorochromes with:

�antigens expressed at low density

�antigens with non-uniform expression

�antigens with unknown expression patterns

• Adjust instrument settings for optimal resolution

of targets

Defining bright versus dim: Stain IndexDefining bright versus dim: Stain Index

Stain Index (SI) = D/W

D = difference between pos and neg peak medians

W = 2 * robust SD

Relative brightness of fluorochromes Relative brightness of fluorochromes

using CD4using CD48-peak beads in:

Combining detector & fluorochrome Combining detector & fluorochrome

performance: assessment of dim antigensperformance: assessment of dim antigens

FITC detector PE-Cy7 detector APC-eFluor® 780

detector

eFluor® 450

detector

CD4 FITC CD4 PE-Cy7 CD4 APC-eFluor® 780 CD4 eFluor® 450

PBMC stained for CD4:

Optimizing instrument settings:Optimizing instrument settings:

PerCPPerCP--Cy5.5 & PECy5.5 & PE--Cy7 exampleCy7 example

0

200

400

600

800

1000

1200

350 450 550 650 750 850

Flu

ore

sce

nce

In

ten

sity

(arb

itra

ry u

nit

s)

Emission Wavelength (nm)

PerCP-Cy5.5 PE-Cy7

Optimizing instrument settings for B220 Optimizing instrument settings for B220

PerCPPerCP--Cy5.5 & CD3 PECy5.5 & CD3 PE--Cy7Cy7

CD

3 P

E-C

y7

B220 PerCP-Cy5.5

CD3 PE-Cy7

B220 PerCP-Cy5.5

CD

3 P

E-C

y7

B220 PerCP-Cy5.5

B220 PerCP-Cy5.5 single stain

PerCP-Cy5.5 detector

626 volts

PerCP-Cy5.5

spillover in the

PE-Cy7 detector

669 volts

626 volts 520 volts

CD3 PE-Cy7

669 volts

520 volts

80%

12%

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• Design





• Nanocrystal considerations


• Analysis



Use Use FluorPlanFluorPlan™ to determine ™ to determine

fluorochromefluorochrome optionsoptions


FluorPlanFluorPlan™ Spectra Viewer™ Spectra Viewer

2011 | www.eBioscience.com

FluorPlanFluorPlan™ Spectra Viewer™ Spectra Viewer



• Design






• Analysis



Using tandem dyes in multicolor flow Using tandem dyes in multicolor flow


• Tandem Dye Consideratons

• Loss of FRET efficiency

�Fixation – use freshly prepared, high quality

formaldehyde

�Light exposure

• Shelf life

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Fixation & Fixation & photostabilityphotostability of PEof PE--Cy7Cy7

Freshly stained

& analyzed cells

Fixed cells:

2% Formaldehyde

30 minutes

Room temp

Fixed cells:

2% Formaldehyde

overnight

4⁰C

Live cells:

Ambient light

6 hours

Room temp

PE detector

Fixation & Fixation & photostabilityphotostability of APCof APC--eFluor® 780eFluor® 780

Fixed cells:

2% Formaldehyde

30 minutes

Room temp

Fixed cells:

2% Formaldehyde

overnight

4⁰C

Live cells:

Ambient light

6 hours

Room temp

APC detector

Freshly stained

& analyzed cells

Additional tandem dye considerationsAdditional tandem dye considerations

• Vendor specific manufacturing protocols

• Compensation requirements can vary

between conjugations (different antibodies)

and even different lots of the same antibody

• Compensation for tandem dye conjugates

should ALWAYS be set with the antibody used

in the staining panel



• Design







• Analysis



Designing a multicolor flow cytometry Designing a multicolor flow cytometry

experimentexperiment

• Minimize spillover

• Consider compensation issues when assigning

fluorochromes to antigens

�Avoid spillover from a bright population into a

detector requiring high sensitivity

• Optimize the core markers of your staining panel for

best resolution



• Design







• Analysis



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Analyzing a multicolor flow cytometry Analyzing a multicolor flow cytometry


• Singlet gating and excluding dead cells

• Critical for the elimination of false positives

• Appropriate controls for gating

• FMO (Fluorescence Minus One)

• Isotype controls

• Internal negative

Principle of singlet gating


Sig

na

l in

ten

sity

time

Single-cell

laser

Sig

na

l in

ten

sity

time

Doublet

laser

Practice of singlet gatingPractice of singlet gatingCD3+ CD19+ double-positive cells?

Value of singlet gatingValue of singlet gating

No! Just 2 cells stuck together.

Dead cells interfere with optimal Dead cells interfere with optimal

staining resolutionstaining resolution

2011 | www.eBioscience.com

Fresh thymocytes

Anti-CD3-stimulated thymocytes

96 2011 | www.eBioscience.com

SingletsViable

Eliminating false positives with singlet Eliminating false positives with singlet

gating & exclusion of dead cellsgating & exclusion of dead cells

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Analyzing a multicolor flow cytometry Analyzing a multicolor flow cytometry


• Singlet gating and excluding dead cells

• Critical for the elimination of false positives

• Appropriate controls for gating

• FMO (Fluorescence Minus One)

• Isotype controls

• Internal negative

Negative spreading phenomenonNegative spreading phenomenon

Fluorescence Minus One (FMO)Fluorescence Minus One (FMO)

• In multicolor experiments, compensation can

introduce error, resulting in “negative

spreading”

• The FMO control is a sample containing every

fluorochrome-conjugated Ab but one

• Including appropiate reference e.g. Isotype

control


CO

MP

EN

SA

TIO

N

CO

MP

EN

SA

TIO

N

Isotype controls: Ubiquitous…Isotype controls: Ubiquitous…

• An Ab of the same isotype conjugated to the

same fluorochrome

Rat IgG2a PE

Human PBMC were stimulated for 5 hrs

with PMA & ionomycin in the presence

of brefeldin. CD3+CD4+ cells were used

for analysis

Autofluorescence (lymphocytes)

Rat IgG2a PE (isotype)

Unstimulated cells

Stimulated cells

IL-2 PE

Limitations of the isotype controlLimitations of the isotype control

• It is its own protein

– Although it is the same isotype, it is still a completely different Ab molecule

• Separate conjugation reaction

– F/P can vary slightly from reaction to reaction

• Isotypes were originally generated & evaluated for non-binding to surface proteins

– How they behave intracellularly can be unpredictable

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Multicolor flow cytometry: SummaryMulticolor flow cytometry: Summary

• Know your instrument capabilities & adjust settings to optimize performance

– Minimize compensation

– Maximize stain index

– Be aware of negative spreading

• Choose markers & fluorochromes to maximize stain index for each marker

– May take a couple tries for more complex panels

• Eliminate false positives (singlets & dead cells)

• Include relevant controls (isotype, FMO, etc.)

Apoptosis, Necrosis, Viability, Vitality, Functionality.......

Apoptosis from A to Z

105eBioscience Confidential | eBioscience.com

Apoptosis

• Greek: apo – from, ptosis – falling

• One of the main types of Programmed Cell Death (PCD)

• Multi-biochemical event (incl. cell to cell interaction)

• Leading to cell death; save dispose of the cell

• In contrast; Necrosis is uncontrolled cell death from acute cellular injury

• On going increase in Apoptosis research since the early ’90

• Many focus areas such as uncontrolled proliferation => Cancer

• As an example, Apoptosis is needed in the differentiation of fingers during ..development of the embryo

~50 to 70 billion of cells undergo apoptosis every day

(average human adult)

In one year the mass of the cells undergoing apoptosis is

~equal to the weight of the individual body


Apoptosis vs Necrosis


Induction and Regulation of Apoptosis

Induction

Cell repair (e.g. DNA damage due ionizing radiation )

Infection (e.g. v irus)

Cell stress (e.g. starvation)

Homeody namics (homeostasis)

Control:

Cell signals

Hormones

Grow th factors

Cy tokines

Nitric ox yde

Regualtion

cell-cell interaction (e.g. haematopoesis)


Apoptosis Inducers in Cell Biology

Actinomycin DCamphtothecin

CycloheximideDexamethasone

Etoposide

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109eBioscience Confidential | eBioscience.com 110eBioscience Confidential | eBioscience.com

Normal Cell

Apoptotic Body (AB) Formation

Lysis of AB

Continued Blebbing

Cell shrinkage Chromatin Condensation

Membrane Blebbing

Nuclear Collapse

The Apoptosis Process


Apoptosis induced Cell function changes

Mitochondrial

Transition Pore opening

Phosphatidyl

Serine (PS) Translocation

Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB


Mitochondrial membrane potential

• JC-1 is a membrane permeable dye for flow cytometry and fluorescent microscopy.

• Selectively enters the mitochondria where it reversibly changes color as membrane potentials .. ..

..increase (over values of 80-100 mV).This property is due to the reversible formation of J-aggregates upon membrane polarization.

• Excitation: 488 nm @ 59%

498 nm @ 82%

592 nm @ 100%

• Emission (max): 530 nm (JC-1 monomer)

590 nm (J-aggregate)

• JC-1 is both qualitative, in regards to the shift from green to orange fluorescence emission, ..and.quantitative, as measured by fluorescence intensity, in both filter sets.

JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide)



Mitochondrial


Phosphatidyl


Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB

Cytochrome C Release


Cytochrome C detection

Cytochrome C Release

MitochondrialTransition Pore

opening

MitochondrialMembrane Potential

Human Cytochrome c Platinum ELISA

Cytochrome C detection via MonoclonalAntibodies

� Flow Cytometry

� Western Blot

� Immuno Histochemistry

� Imaging

� Immuno Precipitation

� Functional Assays

� ELISA

� Human� Mouse� Rat

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Fura-2 AM• Preferred dye for ratiometric imaging microscopy with digital image analysis• Upon binding Ca2+, the excitation spectrum of Fura-2 shifts to shorter

..wavelengths (400 to 300 nm)

• Peak emission remains steady (~510 nm)• Peak excitation: depending on free Ca2+conc. between 300nm and 400nm

• Peak emission: 510 nm• Molecular weight: 1001.86 Da

Indo-1 AM• single excitation / dual emission dye• Unbound Indo-1 has a peak emission at 485 nm, which shifts to 410 nm

upon Ca2+ binding. In Flow Cytometry, this shift can be measured over time

and represented as a ratio of the two emission wavelengths.• Peak Excitation: 346 nm

• Peak Emission: 475 nm (Unbound Indo-1 =485nm shift to 410nm dep. on Ca2+ binding)

• Molecular Weight: 1009.91

Calcium Sensor Dye eFluor® 514• Indicator for intracellular free calcium

• Detection by: FC, IHC, Imaging, Microplate Readers• Increased cellular uptake � increased brightness

• Excitation: 488 nm

• Emission: 514 nm• Calcium binding affinity: Kd = 232 nM

• MW: ~1100 Da

• Not recommended for quantitative measurements

Calcium Sensor Dye eFluor® 514

Jurkat cells were harvested, w ashed and loaded with

Calcium Sensor D ye eFluor® 514 for 30 minutes at 37°C.The left panel shows c ells that were w ashed and analyzed

by flow cytom etry unstimulated (b lue histogram) or

stimulated with 1 ug/mL ionomycin (purp lehistogram).The

right panel shows Jurkat cells loaded with Calcium Sensor

Dye eFluor® 514 that were acquired on a flow cytom eterfor 1 minute and then removed for theaddit ion of 1 ug/mL

ionomycin and immediately p laced back on the flow

cytometer for continued acquisit ion..

Calcium Sensing Dyes



Mitochondrial


Phosphatidyl


Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB

Phosphatidyl Serine (PS) Translocation


Untreated Treated

10µM Camptothecin, 4h

Detection of Phosphatidyl serine (PS) translocationvia Annexin V labeling

Cell M

em

bra

ne

CytosolPhosphatidyl serine

Control:

Counterstain with cell

impermeant DNA-dyee.g. PI, 7AAD

Normal C ell Early Apoptosis Late Apoptosis Necrosis

Formats@eBioscience:

eFluor 450

FITCR-PE

PerCp-eFluor 710

APC

PE-Cy7

Biotin


Annexin V FITC

&

Propidium iodid

Etopodise treated

Thymocytes

MAG 40x MAG 100xMAG 40x

Detection of Phosphatidyl serine (PS) translocationvia Annexin V labeling



Mitochondrial


Phosphatidyl


Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB

CaspaseActivity


Caspase activity

C aspase 1 (pc) H WB, IP, IHC

C aspase 2L p12 H,M,R WB, IP, IHC

C aspase 3 (pro) M WB

C aspase 3 (pc) H WB, IP, IHC

C aspase 7 H WB

C aspase 8 H WB

C aspase 10 H WB

C aspase 11 M FC

C aspase 12 H,M,R WB

C aspase 12 M WB, IP, IHC, FC

C aspase 13 H,M,R WB

WB: Western Blotting

IP: Immuno Precipitation

IHC: Immuno His to Chemis try

FC: Flow Cytometry

H: Human

M: Mouse

R: Rat

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CaspGLOW™

• Detection of active Caspases in live cells• Caspase inhibitors are conjugated to FMK

� provide cell-permeability� non-toxic

� irreversible binding• Detection in Flow Cytometry and Imaging• One hour procedure

Data provided by:

Simple Protocol

Collect cells and resuspend in

incubation buffer

Add caspase probe and Incubate

@37C for 20min

Analyze



Mitochondrial


Phosphatidyl


Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB

MetabolicActivity


Metabolic activity (Viability dyes)

• Calcein is a green fluorescent viability dye excited @ 488nm • Acetoxymethyl ester (AM) makes the molecule hydrophobic and suitable for ..uptake across cell membranes in live cells• Calcein AM becomes highly fluorescent when the acetoxymethyl ester is..hydrolyzed by cytoplasmic esterases thereby releasing the hydrophilic highly ..fluorescent cell-bound calcein• Since dead cells do not contain active esterases, dead cells are not labeled• Calcein AM is not toxic so it can also be used for short-term cell tracing studies

• Calcein Blue AM is a UV excited alternative to Calcein AM, having an excitation ..similar to DAPI, Hoechst, and AMCA

• Calcein Violet 450 AM, is a violet laser (405 nm) excited equivalent to Calcein ..AM. Co-staining with Annexin V or 7-AAD is recommended to allow the greatest ..resolution between live and dead/ apoptotic cells

• Propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are both ..fluorescent viability dyes that can be used to measure plasma membrane ..integrity; only cells with compromised plasma membranes will stain with PI and ..7-AAD


Metabolic activity (Cell Tracking and Proliferation)

Cell Tracking & Proliferation Dye

• CFSE [5-(and 6)-C arboxyfluorescein diacetate succinimidyl ester]

• C ell tracking and proliferation studies

• Also used in CTL assays and cell motility studies

• C FSE readily crosses intact cell membranes, inside the cells, intracellular esterases cleave the

..acetate groups to yield the fluorescent carboxyfluorescein molecule. The succinimidyl ester

..group reacts with primary amines, crosslinking the dye to intracellular proteins.

• Cell division can be measured as successive halving of the f luorescence intensity of .. ..

. C FSE




• CPD eFluor™670: Cell Proliferation Dye eFluor™ 670• Cell tracking and proliferation studies• Also used in CTL assays and cell motility studies• Excitation 633nm; Emission 670nm (APC Filter 660/20 BP)• Works similar than CFSE• Compatible with GFP and YFP detection• For in vivo and in vitro applications




• CPD eFluor™450: Cell Proliferation Dye eFluor™ 450• Cell tracking and proliferation studies• Also used in CTL assays and cell motility studies• Excitation 405nm; Emission 450nm (450/50 BP)• Works similar than CFSE• Compatible with most reporter proteins• For in vivo and in vitro applications

• Mouse splenocytes • 10 uM CPD eFluor® 450

• cultured for 3 days

• with ConA (blue histogram) • without ConA (purple histogram)

• Splenocytes (Thy1.1 mice) • 10 uM CPD eFluor® 450

• Injected into C57Bl/6 mice (purple histogram)

• Injected into B6D2F1 mice (blue histogram)• Splenocytes collected 72 hours after injection

• Unlabeled cells (Gray)

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Cell Proliferation detection via BrdU

BrdU(Bromodeoxyuridine)

• Detection of proliferation (S-phase)

• BrdU is a synthetic nucleoside analog of Thymidin• Thymidin will be replaced by BrdU during replication

Simplif ied

Protocoll

Safes up to an 1h

time compared to

conventional

methods


Simultaneous Measurement combining Proliferation Dyes:

CFSE or Cell Proliferation Dyes

Immunophenotyping; surface markers

Annexin V

Live/Dead discrimination;7AAD, FVD

Nuclear stains; BrdU, transkription factors

Cytokine expression

The staining is always depended on the cell type. Fixation and Permeabilisation can effect performance. Some buffer might effect performance.



Mitochondrial


Phosphatidyl


Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB

P lasmamembrane Integrity


Plasma membrane integrity

life/dead discrimination using cell impermeant dyes

Mouse t hymocytes were prep ared as a single ce ll

suspensio n a nd incubated over night at 37 °C inmedium (left ) or medium wit h 1µM de xamet hasone(right). Cel ls wer e harvested and sta ine d us ing the

Annexin V FIT C Apopt osis Detect ion Kit and Prop id iumIodide Staining Solution (cat. 00-6990).

Propidium Iodide (PI)

• Exclusion of nonviable cells in flow cytometric analysis

• Binds to double stranded DNA by intercalating between ..basepairs

• Is excluded from cells with intact plasma membranes

• Can be used in FL3 for inviability exclusion, but should be ..analyzed in FL2 when used as a counterstain for FITC

..Annexin V.

7-amino-actinomycin D (7-AAD)

• Exclusion of nonviable cells in flow cytometric analysis.

• Can be used in place of PI (propidium iodide) to reduce ..wavelength spill over in Multicolour experiments

• Can be used in combination with PE (phycoerythrin) and

..FITC (fluorescein isothiocyanate) conjugated antibodies • Fluorescence is detected in the far red range of the

..spectrum (650 nm long-pass filter).

7AAD stain o n human L ymphocytes after f reeze/t haw

cycle

7AAD

15%


Life/Dead discrimination; Fixed Cells

Fixable Viability Dyes are a viability dyes that can be used to irreversibly label dead cells prior to:

� Cryopreservation

� Fixation

� Permeabilization

Unlike 7AAD and propidium iodide, cells labeled with Fixable Viability Dyes can be washed, fixed,

permabilized, and stained for intracellular antigens without any loss of staining intensity of the dead

cells. Thus, using Fixable Viability Dyes allows dead cells to be excluded from analysis when intracellular targets are being studied. Fixable Viability Dyes may be used to label cells from all species.

Fixable Viability Dye eFluor® 450





Amine-reactive fluorescent reagents Binds free amine groups on proteins

Unlabeled cells

Labeled cells� Intact membrane

Labeled cells� Compromised membrane

NO

fluorescence

MEDIUM

fluorescence

BRIGHT

fluorescence

Life/ Dead discrimination; Fixed Cells

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Amine-reactive fluorescent reagents Binds free amine groups on proteins

NO

fluorescence

MEDIUM

fluorescence

BRIGHT

fluorescence

Life/ Dead discrimination; Fixed Cells






Nuclear Labeling of DNA

�anthraquinone dye

�high affinity for double-stranded DNA�membrane-permeable dye

�nuclear DNA content analysis eg. for ploidy /cell cycle analysis because it binds DNA stoichiometrically� In fluorescent microscopy, it can be used as a nuclear counterstain.

For cell cycle/DNA analysis applications in order to optimize to optimize the CV for the G1 and G2/M peaks a higher wavelength filter is recommended:

710LP, 735LP dichroic mirror and 780/60 band pass),

Do not combine with far-red fluorochromes excited by the 488 or 633 nm laser lines

(PE-Cy7, PerCP-eFluor® 710, PerCP-Cy5.5, APC, Alexa Fluor® 647, Alexa Fluor® 700, APC-eFluor® 780)

Cat. 65-0880

Nuclear Red (DRAQ 5™)

Ex: 488-647nm

Ex: 488nm

Detect: P erCP-eFluor® 710 or P erCP-Cy5.5

(685LP dichroic mirror and 710/50 BP)

Ex: 633nm

Detect: APC (660/20 band pass)C57Bl/6 bone marrow cells were stained with FITC anti-mouse CD45 (30-F11) (cat.

11-0451) (left) and eFluor® 450 anti-mouse TER-119 (cat. 48-5921) (right), followed by staining with 5 µM Nuclear RED (DRAQ5™) for 15 minutes at room temperature. Total viable cells were used for analysis.


Nuclear Labeling of DNA

� anthraquinone dye

�high affinity for double-stranded DNA

�membrane-permeable dye

� In flow cytometry, it can be used to distinguish nucleated and non-nucleated cells

� In fluorescent microscopy, it can be used to identify and discriminate the nucleus and cytoplasm

…without the need for a second dye due to its high intensity staining of the nucleus and low intensity

…staining of the cytoplasm.

Please make sure that your instrument is capable of detecting this dye.

Cat. 65-0881Nuclear ORANGE (CyTRAK Orange™) Ex: 488-550nm Ex: 488nm Em max: 610nm (610/20 BP)


Cell Tracking; CellVue® dye

�lipophilic dyes

�labeling of the cell membrane

�identifying and tracking of labeled cells

�rapid and stable labeling

�Multiplexable

�Supplied with diluent and labeling vehicle

C ellVue® Lavender (Cat.88-0873) Ex: 420 Emmax: 461

C ellVue® Jade (Cat.88-0876) Ex: 478 Emmax: 508

C ellVue® NIR780 (Cat.88-0875) Ex: 633 Emmax: 776

C ellVue® Maroon (Cat.88-0870) Ex: 647 Emmax: 667

C ellVue® Plum (Cat.88-0871) Ex: 652 Emmax: 671

C ellVue® Burgundy (Cat.88-0872) Ex: 683 Emmax: 707

C ellVue® NIR815 (Cat.88-0874) Ex: 786 Emmax: 814



Mitochondrial


Phosphatidyl


Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB

DNA Fragmentation


• Detection of apoptotic cells by flow cytometry• 2-color staining method• Labeling DNA breaks and total cellular DNA

The kit content:

� Instructions, all reagents required, positive and negative control� Washing, reaction and rinsing buffers� Terminal deoxynucleotidyl transferase enzyme (TdT)� Fluorescein-deoxyuridine triphosphate (dUTP)� Propidium iodide / RNase A solution for counterstaining the total DNA

One of t he most easil y measured features of apoptotic cells is t he break-up of t he genomic DNA by cell ular

nucl eases. These DNA fragments c an be extract ed from apoptotic cells and res ult in t he appearance of DNAladderi ng w hen t he DNA is anal yzed by agarose gel electrophoresis. The DNA of non-apopt otic c ells t hat

remains largel y i ntact does not displ ay t his l adderi ng on agaros e gels duri ng el ectrophoresis. The large

number of DNA fragments appearing in apoptotic cells results in a multitude of 3' -hydroxyl termini i n t he

DNA. This propert y can be used to identi fy apoptotic cells by l abeling t he 3'-hydroxyl ends with directly

conjugated fluorescein- deoxyuri di ne tri phosphat e nucleotides (FITC-dUTP). The enzyme terminaldeoxynucl eotidyl trans ferase (TdT) catal yzes a templat e-i ndependent addition of deoxyribonucleoside

triphosphat es to the 3'-hydroxyl ends of double- or si ngle-stranded DNA wit h eit her blunt, recessed or

overhanging ends. A substantial number of t hese sites are available i n apoptotic cells providing the basis for

the method utiliz ed in the APO-DIRECT™ Kit. Non-apoptotic c ells do not inc orporat e signi ficant amounts of

the FITC-dUTP due to the lack of exposed 3'-hydroxyl DNA ends.

DNA break vs total DNA: APO-DIRECT™

APO-DIRECT is a trademark of Phoenix Flow Systems, San Diego, California.

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• Anti-ssDNA stain of condensed chromatin• Staining of cells and/or tissue sections with• APOSTAIN followed by heat treatment, induces DNA denaturation in situ only in..apoptotic nuclei.• In the presence of formamide, only apoptotic nuclei DNA becomes denatured and..detectable• Applicable for Human, Mouse and Rat samples• For Flow Cytometry• For IHC staining including formalin fixe, paraffin embedded tissue sections

APOSTAIN™

DAPI counterstain APOSTAIN positive nuclei



Mitochondrial


Phosphatidyl


Caspase Activity

Metabolic Activity

DNA

Condensation

Plasmamembrane

Integrity

DNA

Fragmentation

Normal Cell

Lysis of AB

Mitochondrial

Membrane Potential

Normal Cell

Lysis of AB



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…..perfectionperfectionperfectionperfection isisisis finallyfinallyfinallyfinally attainedattainedattainedattained

notnotnotnot whenwhenwhenwhen theretheretherethere isisisis nononono longerlongerlongerlonger anythinganythinganythinganything totototo add,add,add,add,

butbutbutbut whenwhenwhenwhen theretheretherethere isisisis nononono longerlongerlongerlonger anythinganythinganythinganything totototo taketaketaketake

awayawayawayaway ............

Antoine de Saint Exupéry (1900-1944), Terre des Hommes (1939)


Thank You

FlowCytometry 2013-Handout

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