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Transcript of Cellutions V12011 EMD Millipore
EMD Millipore is a division of Merck KGaA, Darmstadt, Germany
CellutionsVol 1: 2011
PaintYour Nuclei
Live Imaging-Based Chemical Screens Using Neural Stem Cells page 8
Rapid Plate-Based Cytometric Methods for Mitochondrial Screening page 10
New Chemotaxis Assay for Single Cell Analysis Using a Microscale Migration Chip page 16
The New Scepter™ 2.0 Cell Counter Enables the Analysis of a Wider Range of Cell Sizes and Types With High Precision page 19
To subscribe to the quarterly Cellutions newsletter,please visit www.millipore.com/cellquarterlynews
A Dual Detection Approach Using H2A.X for Measuring DNA Damage by Flow Cytometry page 3
The Newsletter for Cell Biology Researchers
2
With the new easyCyte™
single-sample flow cytometer,more = less.See what our new FlowCellect™ assay kits, easyCyte instruments, and
InCyte™ software can do — read our research articles on pages 3 and 10
of this issue of Cellutions!
Visit www.millipore.com/flowcytometry to learn more and request a demo.
easyCyte 8 Features
• Microcapillary flow cell requires no sheath
fluid and is user-replaceable
• Up to six-color detection
made possible by one (blue) or
two excitation lasers (blue and red)
• Small footprint saves valuable
laboratory space:
Width: 17.75 in (45.1 cm)
Depth: 17.25 in (44.5 cm)
Height: 8.75 in (22.2 cm)
(does not include laptop)
• Single sample loader Swivel arm functionality, holds two tubes
and allows instant acquisition
• Waste vial collects less than 80 mL of
waste in a typical 8-hour workday
• Wash vial offers a
high-pressure purge to easily clear
obstructions from the flow cell
prodUct HIGHLIGHt
+ MORE PARAMETERS
+ MORE ANALYTICS
+ MORE INSIGHTFUL DATA
+ MORE BENCHTOP SPACE
+ MORE SIMPLICITY
+ MORE SOLUTIONS
= LESS $$$
3
AbstractInvestigating the DNA damage pathway is
important for understanding genome health
and cancer. Defects in DNA damage pathways
can lead to genetic instability, uncontrolled
cell growth, and, ultimately, tumorigenesis.
Proliferating cells are especially vulnerable to
DNA damage due to the added stress placed on
DNA by cellular growth and division processes.
Having a comprehensive understanding of
DNA damage can be critical to
interpreting the intrinsic nature
of cell proliferation, apoptosis,
and DNA repair and can assist
in the development of anti-
neoplastic agents. Here, we
describe the development of an
assay for quantitation of DNA
damage in individual cells and
the use of this assay to screening
small molecules for their
potential to cause DNA damage.
Our novel, dual detection assay
platform uses pairs of total and
phospho-specific antibodies
for multi-color flow cytometry
analysis. We selected a pair of
H2A.X antibodies that bind to
the same protein target: one to
detect total H2A.X expression
and another to detect the
phosphorylated form of the
same protein. Simultaneous
analysis of total and
phosphorylated H2A.X provides
accurate quantitation of DNA
damage in a cell sample, because
it can confirm target specificity
of the phosphorylation event,
even in a mixed cell population. Our data
indicate that the use of total/phospho antibody
dual detection flow cytometry is an effective
approach for studying the extent of DNA
damage and screening of kinase inhibitors.
IntroductionHistone H2A.X resides downstream of the
DNA damage kinase signaling cascade.
Phosphorylation of Histone H2A.X at serine
139 is an important indicator of DNA damage1.
As the level of DNA damage increases, the
level of phospho Histone H2A.X (also known as
gH2AX) increases, accumulating at the sites of
DNA damage. This accumulation of phospho
Histone H2A.X is often used to indicate the
level of DNA damage present within the
cell1. H2A.X is also responsible for recruiting
response proteins to the site of DNA damage
and may play a role in DNA repair2.
DNA content levels vary with respect to
stages of the cell cycle, and histone doubles
in content during the cell cycle at the same
rate DNA content doubles1. To compensate for
this increase, we developed a multiplexed flow
cytometry assay for both total and phospho
H2A.X. By staining cells with antibodies
recognizing both total and phospho H2A.X., we
were able to normalize the amount of phospho
H2A.X detected to changes in DNA content in
cycling cells.
Ionizing radiation (IR) and many
chemotherapeutic agents like etoposide
kill cancer cells by induction of DNA DSBs.
Several reports show that the level of gH2AX
as detected by flow cytometry correlates
with the number of DNA strand breaks, to
A Dual Detection Approach Using H2A.X for Measuring DNA Damage by Flow CytometryMark Santos, Kevin Su, chintya Ganda, roberto renteria, Jason Whalley, and Matthew Hsu
MRNComplex
Ionizing Radiation
Changes inChromatin Structure
ATM
H2AX
SMC1
53BP1 MDC1
p53
Apoptosis
DNA RepairCell-cycleCheckpoint
Arrest
ATM
ATM
BRCA1
CHK2
P
PP
PP
PP
PP
PP
PP
4
the level of cell death and radiosensitivity3.
H2A.X is phosphorylated in response to a DNA
damaging reagent (e.g. Etoposide) or UV light,
and its activation clearly indicates that DSBs
have occurred. Understanding when DSBs
take place can help researchers understand the
mechanisms involved in DNA repair and the
DNA damage response.
In this study, we evaluated the effects of
etoposide treatment on HeLa cells. DNA
topoisomerase inhibitors induce lethal
chromosome damage, including breaks and
rearrangements4. After stimulating HeLa cells
with etoposide for 2 hours, we detected a
marked increase in phosphorylated H2A.X.
To further investigate the effect of anti-
neoplastic agents on DNA damage, deep dive
analysis was conducted by titration of various
small molecules to define EC50 values (Figures
2 and 3). First we examined the EC50 value of
one small molecule (etoposide) at two different
exposure times. Then, since structure-activity
relationships (SAR) of small molecules are
critical in identifying selective anti-neoplastic
agents, we measured EC50 values of multiple
molecules with varying structure using the
same flow cytometric assay.
Methodsdual detection of H2A.X: We used the
FlowCellect™ DNA Damage Histone H2A.X
Dual Detection Kit to measure total and
phosphorylated H2A.X. FlowCellect Dual
Detection Kits are a series of flow cytometry
products which include a pair of antibodies
that bind to the same protein; one to detect
total protein expression and another to detect
the phosphorylated form of the same target.
Using two parameter analysis, we can achieve
target specific detection of phosphorylation
and, by doing so, eliminate false positives while
enhancing the signal to noise ratio.
To validate the useful application of this kit
by accurately measuring and quantitating
the extent of DNA damage and DNA stranded
breaks, a human cervical carcinoma cell line
(HeLa cells) was used for DNA damage analysis.
Prior to cell staining, cells were first treated
with etoposide (a topoisomerase II inhibitor)
for 2 hours at 37°C. Following treatment, the
cells were immediately fixed and permeabilized
with EMD Millipore’s proprietary fixation and
permeabilization buffers to ensure proper
access for antibody entry into the cell. Cells
were then co-stained with an anti-phospho-
H2A.X (Ser139) PerCP-conjugated antibody and
with an anti-H2A.X FITC-conjugated antibody
for 30 minutes at room temperature in the
dark. A non-treated cell sample was used as
a negative control. Following cell staining,
a series of washing steps were performed to
remove any unbound antibody. Data were
acquired using a guava® flow cytometer and
H2A.X activation levels were determined
using bivariate analysis, plotting total versus
phospho H2A.X.
Small molecule structure-activity relationship (SAr) evaluation using phospho H2A.X: In order to demonstrate that
flow cytometry is a viable tool to complement
any screening campaign, we used a phospho-
specific antibody as a SAR tool for compound
evaluation. HeLa cells were pretreated with
etoposide for 2 hr and 24 hrs at 37°C in a
half log, dose dependent manner. Following
treatment, cells were then fixed, permeabilized,
and stained. The mean fluorescence values,
or MFI, were then determined and plotted
using a curve-fitting algorithm built into
EMD Millipore’s InCyte software module to
construct EC50 dose response curves. To rank
order compounds, various small molecules
indicated to induce DNA stranded breaks were
administered to HeLa cells in varying doses.
Twelve-point half-log serial dilutions were
performed, and EC50 values were derived from
dose response curves generated using the
InCyte software module for curve-fitting. Cells
were pretreated with the compound of interest
(figures #2 and #3) for 24 hours prior to cell
fixation, permeabilization, and cell staining.
resultsBy performing bivariate analysis using both
an anti-phospho-H2A.X and anti-H2A.X (total)
antibodies in multiplex, not only were we
able to measure the extent of DNA damage
with great accuracy and confidence, but we
also achieved target-specific detection of
phosphorylation, eliminating false positives
while enhancing the signal to noise ratio.
By plotting total H2A.X on the X-axis against
the phospho-specific version of the same
protein on the Y-axis of a bivariate dot plot,
A. Unstimulated B. Stimulated
H2AX-FTC (GRN-HLog)
Total H2AX - FITCPh
osph
o H2
AX -
Per
CP
pH2A
X-Pe
rCP
(RED
-H_c
g)
H2AX-FTC (GRN-HLog)
pH2A
X-Pe
rCP
(RED
-H_c
g)
TWO PARAMETER ANALYSIS USING TOTAL & PHOSPHO ANTIBODIES
Figure 1. DUAL PARAMETER ANALYSIS OF TOTAL AND PHOSPHO HISTONE H2A.X ON HELA CELLS Unstimulated HeLa cells stained with both phospho-Histone H2A.X-PerCP and Anti-Histone H2A.X-FITC (A) showed no indication of Histone H2A.X activation via phosphorylation. Total H2A.X was detected in 97.2% of cells. However, once HeLa cells were stimulated with 100 µM etoposide, simultaneous measurement of both total and phospho Histone H2A.X confirmed H2A.X-specific phosphorylation population (B) as indicated by the 2.09% to 97.15% increase in double positive staining
5
PAINT YOUR NUCLEI
6
we only detected cells expressing H2A.X in
unstimulated samples (cells marked in Figure
1A by the shift in cell population, 97.22%).
Because these cells were not treated with a
DNA damage inducer, there was no activation
of DNA damage response, as indicated by the
lack of cells containing phospho-H2A.X (2%,
Figure 1A). However, upon treatment with
etoposide for 2 hours, H2A.X activation was
manifested by the upward shift of cells into
the upper right quadrant of the dot plot (from
2% to 97% of cells, Figure 1B). This indicated
that DNA damage had occurred, resulting
in the phosphorylation of H2A.X. We could
thus measure and normalize the extent of the
damage by implementing this dual detection
approach.
As also shown in Figure 1, the total H2A.X
protein level remained constant compared to
unstimulated cells, while phospho-Histone
H2A.X levels increased in all cells, indicating
that there was no competition between the
two antibodies for their target epitopes.
This absence of interaction or competition
suggested that this dual detection method
could be an attractive and sensitive assay for
quantifying double-stranded DNA breaks.
Structure-activity relationships for drug screeningHaving the ability to compare and rank-order
compound activity is crucial for many drug
screening campaigns. We demonstrated
a flow cytometry method for comparing
various compound activities based on the
mean fluorescence intensities generated by
increasing binding of fluorescently conjugated
anti-phospho-H2A.X to cells titrated with
DNA damage inducers. As shown in figure
2, etoposide was serially diluted and applied
to HeLa cells at two time points (2 hours and
24 hours) in 12-point, half-log dilutions to
generate EC50 curves. As indicated by the
EC50 values calculated from the dose response
curves, longer incubation times appeared to
influence small molecule activity and efficacy.
This information could be useful in identifying
advanced small molecule leads. In figure 3,
various small molecules were rank-ordered by
flow cytometry, further illustrating how flow
Figure 2. DOSE RESPONSE CURVES FOR THE TOPOISOMERASE INHIBITOR, ETOPOSIDE. HeLa cells were treated with etoposide for either two hours (A), or for 24 hours (B). As illustrated, different incubation times can influence drug activity. This can serve as a useful utility to drive SAR drug screening campaigns.
Figure 3. COMPOUND RANK ORDERING BY FLOW CYTOMETRIC ASSESSMENT OF DNA DAMAGE-CAUSING AGENTS. HeLa cells were dosed with various small molecules for 24 hours prior to flow cytometry using a phospho-specific H2A.X –PerCP antibody. Compounds were rank-ordered based on their efficacy as measured by EC50 values derived from dose-response curves.
A. Two-hour incubation B. 24-hour incubation
EC50 = 38 µM EC50 = 9.4 µM
A. Topotecan B. Anisomycin
C. Aphidicolin D. Etoposide
EC50 = 120 nM EC50 = 10 µM
EC50 = 15 µM EC50 = 9.4 µM
*COMPOUND RANK ORDER: topotecan > Etoposide > Anisomycin > Aphidicolin
7
cytometry could be used as a viable screening
tool to complement any drug screening
campaigns.
discussion & conclusionAs illustrated, we have developed an optimized
assay protocol for evaluating DNA damage
and the ability to measure the degree of
DNA stranded breaks in a given population
of cells. We implemented a dual detection
approach, using an anti-phospho-H2A.X
PerCP-conjugated antibody multiplexed with
an antibody to measure the total levels of the
same protein. The levels of both the total and
phosphorylated protein could be measured
simultaneously in the same cell, resulting in
a normalized and accurate measurement of
H2A.X activation after stimulation. Moreover,
simultaneous measurement of both total and
phospho-Histone H2A.X confirmed target
specificity of the phosphorylation event. In
general, a total and phospho antibody duo
applied in multiplex provides an enhanced and
more reliable detection of the phospho: total
protein ratio within a mixed cell population.
We have demonstrated that, using this dual
detection method, we were able to detect the
extent of DNA damage accurately after HeLa
cells are pretreated with etoposide, as was
expected given etoposide’s DNA damaging
properties. We also demonstrated that this
method could facilitate SAR studies by
generating EC50 curves and rank ordering a
select group of compounds. The ability to
accurately measure the extent of DNA damage
while simultaneously performing deep dive
analysis of small molecule activity can provide
researchers a powerful tool in studying disease
states involving DNA damage and repair and
can assist in drug screening, particularly for
cancer therapeutics.
RELATED PRODUCTS
description catalogue No.
FlowCellect DNA Damage Histone H2A.X Dual Detection, 25 tests FCCS025153
FlowCellect Multi-Color DNA Damage Response Kit, 25 tests FCCH025104
FlowCellect MAPK Activation Dual Detection Kit, 25 tests FCCS025106
FlowCellect EGFR RTK Activation Dual Detection Kit, 25 tests FCCS025107
FlowCellect PI3K Activation Dual Detection Kit, 25 tests FCCS025105
FlowCellect Cell Cycle Checkpoint H2A.X DNA Damage Kit, 25 tests FCCH025142
FlowCellect Cell Cycle Checkpoint ATM DNA Damage Kit, 25 tests FCCH025143
REFERENCES1. Tanaka, T., et al. (2007). Cytometry of ATM Activation
and Histone H2AX Phosphorylation to Estimate Extent of DNA Damage Induced by Exogenous Agents. Cytometry; 71A:648-661.
2. Ewald, B., et al. (2007). H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint. Mol Cancer Ther.; 6(4):1239–48.
3. Muslimovic, A., et al. (2008). An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells. Nat Protoc.; 3(7):1187-93.
4. Kaufmann, W.K., et al. (1996). DNA damage and cell cycle checkpoints. The FASEB Journal.; Vol. 10, 238-247.Nunez R. DNA measurement and cell cycle analysis by flow cytometry. Curr. Issues Mol. Biol. 2001;3(3):67-70.
Available from www.millipore.com.
8
area occupied by cells in the microscopic field
at a determinate time point minus the area
occupied before the addition of compounds.
Well-to-well variation was thereby normalized
during initial plating density. A Z’ factor, which
ranges from 0 to 1, was also used to quantify
the resolving power of the screen5. Compounds
were successfully distinguished as either
cytostatic (Z’ factor > 0.95) or cytotoxic
(Z’ factor > 0.75).
Identifying small molecules that promote self-renewal of NES cellsTwo panels of kinase inhibitors,
InhibitorSelect™ 96-Well Protein Kinase
Inhibitor Library I and InhibitorSelect
96-Well Protein Kinase Inhibitor Library II, were
screened to identify chemicals that optimize
and increase the propagation of NES cultures.
These libraries make up a potent, specific,
pharmacologically active, well-characterized,
and structurally diverse set of 160 compounds.
A final DCV was calculated for every time point
Live Imaging-Based Chemical Screens Using Neural Stem Cellsdanovi d, Falk A, Humphreys p, Smith AG, pollard SM
Wellcome Trust Centre
for Stem Cell Research
University of Cambridge,
Cambridge, UK
IntroductionSmall molecules that modulate stem cell
behavior can be useful laboratory tools, which
enable simpler, and more defined conditions
for expanding or differentiating neural stem
cells. In a recent investigation, we screened 160
kinase inhibitors using an IncuCyte® live, high
content imaging system to determine their self
renewal effects on neuroepithelial stem (NES)
cells1,2 (Figure 1). NES cultures were chosen for
their ability to proliferate as stable cell lines
under fully defined conditions, in the absence
of overt differentiation, in medium containing
EGF and FGF-2 growth factors3,4.
Identifying compounds with cytostatic and cytotoxic propertiesPhase contrast images of each well at 30
minute intervals were captured using an
IncuCyte 96-well live imaging system. Each
frame was quantified according to a Delta
Confluence Value (DCV), which is a measure of
the cell number, and is derived from the total
Figure 1: OVERVIEW OF CHEMICAL SCREENING PLATFORM. IPS-DERIVED NES CELLS ARE USED FOR COMPOUND SCREENS. Delta Confluence Values are obtained from single images of the same field on a multi-well plate (6 x 96 wells) at different time points, before and after the addition of a specific compound. Cytostatic compounds (light gray circle) modulating NES self-renewal can be further investigated for their role in differentiation into specific lineages. Compounds promoting expansion of NES (black circle) can be followed up as therapeutic leads for the expansion of endogenous stem cells.
iPS-derived NES cells
Promoting Expansion
Cytotoxic
0 1 2 3 4Time (days)
160 kinaseinhibitors intriplicate
DMSO6 x 96 well plates
9
and every compound tested in triplicate (see
Fig. 2). Twelve mock-treated wells showed a
final DCV value between 43.4% and 54.7%,
while wells treated with kinase inhibitors
displayed a final DCV value between -10%
(cytotoxic) and 90% (proliferative, pro-self-
renewal effects). Importantly, an EGFR inhibitor
was identified as a cytotoxic compound
(DCV = -4.05).
Among the chemicals showing a positive effect
on the propagation of NES cells, three Rho-
associated protein kinase (ROCK) inhibitors,
Y-27632, ROCK inhibitor IV, and HA 1077
Dihidrochloride, produced the highest DCV
values (91%, 88%, and 86%, respectively).
Y-27632 is a highly potent, cell-permeable
and selective inhibitor of ROCK (Ki = 140
nM), and also inhibits ROCK-II. A second
compound, ROCK Inhibitor IV, inhibits ROCK
with an improved selectivity. Finally, HA 1077
Dihydrochloride inhibits ROCK as well as
protein kinase A, protein kinase G and myosin
light chain kinase.
To validate the self-renewal effects of the
aforementioned ROCK inhibitors on
NES cells, Y-27632 (10 µM) was tested in the
presence and absence of EGF and FGF, both of
which are shown to promote NES proliferation.
Results demonstrated that under each
condition the addition of Y-27632 resulted
in increased NES cell expansion compared to
controls.
SummaryUsing an IncuCyte live imaging system,
we determined the self-renewal effects of
160 kinase inhibitors, three of which are
characterized as potent ROCK inhibitors, on
neuroepithelial stem cells. Further studies will
be needed to establish whether these effects in
vitro are due to increased survival, altered cell
cycle kinetics, adhesion, or through inhibition
of differentiation, all of which are biological
functions of Rho kinases that are relevant
to self-renewal. However, the experiments
clearly highlight the utility of our screening
platform to define chemical tools that promote
expansion of NES cell cultures.
NES cells are valuable tools for chemical
screening in studies of neuroregeneration, and
the assays we describe should enable the rapid
identification of chemicals that can modulate
normal and tumorigenic stem cell behavior.
Figure 2: SCREENING OF 160 KINASE INHIBITORS INCLUDED IN InhibitorSelect LIBRARIES I AND II. Data show Delta Confluence Values, corresponding to the change in relative cell number for twelve mock-treated wells and 160 kinase inhibitors. Three compounds, all affecting Rho kinases, were selected as primary hits for their effect on expansion of NES and are detailed in the top right.
RELATED PRODUCTS
description catalogue No.
InhibitorSelect 96-Well Protein Kinase Inhibitor Library I 539744
InhibitorSelect 96-Well Protein Kinase Inhibitor Library II 539745
Y-27632 688000
ROCK Inhibitor IV 555554
HA 1077 Dihydrochloride 371970
EGFR Inhibitor 324674
REFERENCES1. Danovi, D., et al. 2010. Biochem. Soc. Trans. 38, 1067-1071.2. Ding, S., et al. 2004. Nat. Biotechnol. 22, 833.3. Conti, L., et al. 2005. PloS Biol. 38, e283.4. Sun, Y. et al. 2008. Mol. Cell Neurosci. 38, 245.5. Zhang, J.H., et al. 1999. J. Biomol. Screen 4, 67.
0 1 2Time (days)
Y27632Rho-kinase Inhibitor IVHA 1077 dihydrochloride
Aver
age
delta
con
fluen
ce (n
=3)
3 4100
80
60
40
20
0
-20
12
3
123
Available from www.emdbiosciences.com.
10
cellular sample. In this study, we describe
the use of novel flow cytometry assay kits
for simultaneous and rapid, multiparametric
evaluation of apoptosis, mitochondrial and
cell health markers in compound screening
experiments. InCyte analysis software provided
key benefits towards visualizing the action of
a large chemical library on a large group of
samples.
IntroductionMitochondria are critical cellular organelles
that produce 90% of cellular energy and
control cell survival as a part of apoptosis
regulation. Changes in mitochondria are
implicated in multiple cellular processes, such
as the generation of oxidative stress and
the initiation of caspase and non-caspase
mediated apoptosis1. Under proapoptotic
stimuli, mitochondria undergo several changes,
such as depolarization of the mitochondrial
membrane potential and release of inner
mitochondrial proteins such as cytochrome
c into the cytoplasm which in turn results
in the activation of caspases and full-blown
apoptosis2.
Mitochondrial dysfunction is implicated
in a number of disease processes and in
understanding drug/compound toxicity
effects3. Traditionally, the study of
mitochondrial markers has utilized multiple
platforms which have included long laborious
methods and required large sample sizes.
In this study we demonstrate the power
of simplified assay protocols, plate-based
microcapillary flow cytometry, and heat-
map features of InCyte software to rapidly
identify lead compounds4. Jurkat cells were
treated with over a 100 known cytotoxic
compounds from screening plates and the
Rapid Plate-Based Cytometric Methods for Mitochondrial ScreeningKatherine Gillis, Julie clor, rick pittaro, roberto renteria, Angelica olcott, and Kamala tyagarajan
EMD Millipore
AbstractMitochondria play a crucial role in energy
generation and in the maintenance of
cell health. Mitochondrial dysfunction in
disease and compound treatment has dire
consequences for the cell that can result in
apoptosis, necrosis/cell death, or caspase
independent cell death. Monitoring impact on
mitochondria and related cell health markers,
such as caspases and cell death signals, can
provide greater insights on the mechanism
of action in compound screening programs,
pathway mapping, and understanding
apoptosis.
Plate-based microcapillary cytometric
screening with guava easyCyte HT instruments
and InCyte Software allows for rapid
assessment of mitochondrial health and
provides for more complete information
on the mode of action of compounds by
enabling multiplexed analysis from the same
Extrinsic Pathway Signal
Pro-Caspase 8
Pro-Caspase 9
Cyt c
Cyt cAPAF-1
Smac/DiabloAIF
Endo G
Caspase 8
Caspase 3
APOPTOSIS
FAS
Mitochondria
Apop
toso
me
Nuc
leus
∆ψm MitochondrialPotential Change
Activated Caspase Cascade
ER Stress,DNA Damage,Oxidants
DNA Fragmentation
Chromatin Condensation
BaxBak
t-Bid
IAP
IAP
Bid
Figure 1. As the cell’s control center for energy production and survival, the mitochondrion helps integrate stress signals and pro-apoptotic signals to effect cell death signaling to the nucleus via caspase-dependent and caspase-independent pathways.
11
Multiparameter analysis of membrane potential, apoptosis and cell deathAfter induction, 100 µL of cells were washed
and resuspended in 100 µL of 1X Assay Buffer
HSC. A working solution of MitoSense Red dye
and Annexin V, CF488A in 100 µL 1X Assay
Buffer HSC was prepared and added to the
cells. Samples were incubated at 37 ºC for 15
minutes. After incubation, cells were washed
twice and resuspended in 1X Assay Buffer HSC
and stained with 7-AAD. Plates were then
analyzed on a guava easyCyte HT system.
Intracellular monitoring of cytochrome c releaseAfter induction, 100 µL of cells were washed
in 1X PBS, resuspended in 100 µL of 1X
Permeabilization Buffer containing 0.5%
Fixation Buffer, and were incubated on ice for
10 minutes. After incubation, 100 µL of 2.5X
Fixation buffer was added to the cells and
incubated for 20 minutes at room temperature.
Cells were washed 1X, resuspended in 150 µL
of 1X Blocking buffer, and incubated at room
temperature for 30 min. Following blocking,
10 µL of Anti-Cytochrome c-FITC was added
to each well and samples were incubated for
an additional 30 minutes at room temperature.
After the incubation, 100 µL of 1X Blocking
Buffer was added to each well and samples
were washed. Sample were resuspended in
200 µL of Blocking Buffer and analyzed on the
easyCyte HT system.
Monitoring caspase activityAfter induction, 10 µL of Caspase 9 SR and
10 µL of Caspase 8 FAM was added to each
well and incubated in a 37°C CO2 incubator
for 1 hour. After incubation, samples were
washed twice and stained with 7-AAD. After
10 minutes of incubation at room temperature,
samples were then acquired on the easyCyte
HT system.
Cells were analyzed using the guava easyCyte
HT system instrument platform. Heat map data
were obtained using the InCyte software.
resultsThe ability to obtain multiparametric
information during compound screening is
invaluable to the drug discovery process. In
this study, a range of cytotoxic compounds
were screened with three main assays, the
FlowCellect MitoDamage assay, FlowCellect
Cytochrome c assay and guava Caspase
8-FAM and Caspase 9-SR assays to correlate
mitochondrial health with cellular health and
apoptosis.
The FlowCellect MitoDamage assay provides
simultaneous information on mitochondrial
membrane potential changes, apoptosis
as measured by Annexin V binding to
phosphatidylserine residues on the exterior
of the cell and cell death as assessed by
7-AAD reactivity in a single simplified assay.
Results of this assay showed that treatment
of Jurkat cells with 2,6-dimethoxyquinone
results in reduced Red2 fluorescence of the
cells were further split into multiple plates for
mitochondrial assays some of which include
mitochondrial potential changes, Cytochrome
c release, multiple apoptosis markers and
cell death. These plates were subsequently
analyzed by microcapillary cytometry on the
guava easyCyte 8HT platform. Screening of
the plate data was performed using InCyte
Software. Heat map features of the software
allowed for the quick identification of several
cytotoxic compounds and the type and extent
of mitochondrial perturbations they exhibited,
allowing for pathway assessment.
MethodsA non-adherent human T cell line, Jurkat,
were kept in log phase in complete medium
to stimulate optimal growth. Jurkat cells were
assayed in 96-well round bottom plates. For
screening experiments, Jurkat cells were seeded
at 60,000 cells/well (100 µL total volume) just
prior to induction. Following induction, cells
were harvested and assayed as described.
A panel of 160 cytotoxic, immunosuppressive,
anti-proliferative, and anti-inflammatory
compounds were obtained from Microsource
Discovery Systems, Inc. Vendor supplied
compounds at a concentration of 10 mM
in DMSO. For initial screening experiments,
compounds were diluted into complete growth
medium to 10 µM. The outside columns of the
plates were set up with negative or positive
controls. Negative control wells containing
0.2% DMSO and a positive control wells
contained, 1 µM staurosporine, a known
apoptotic inducer. Plates were induced for
either 4 hours or 24 hours.
For dose response experiments, camptothecin
and gambogic acid were diluted in complete
growth media at the concentrations described.
Cells were induced in Camptothecin for
6 hours at concentrations ranging from
0.3-30 µM while cells in gambogic acid were
induced for 4 hours at concentrations ranging
from 0.15-200 µM.
Following treatments, cells were stained
with FlowCellect MitoDamage, FlowCellect
Cytochrome c, and guava Caspase 8-FAM &
Caspase 9-SR kits.
Figure 2. Treatment of Jurkat cells with 2,6-dimethoxyquinone followed by analysis with the MitoDamage kit showed that treatment with 2,6-dimethoxyquinoine results in decrease in mitochondrial potential, increased apoptosis and cell death under conditions evaluated.
B. 2,6-dIMEtHoXyqUINoNE-trEAtEd JUrKAt cELLS
Mito
Sens
e Re
dAnnexin V, CF488A
Mito
Sens
e Re
d
7-AAD
7-AA
D
Annexin V, CF488A
A. 2,6-dIMEtHoXyqUINoNE-UNtrEAtEd JUrKAt cELLS
Mito
Sens
e Re
d
Annexin V, CF488A
94.4% 1.1%
0.75% 3.7% Mito
Sens
e Re
d
7-AAD
95.2% 0.3%
3.2% 1.3%
7-AA
D
Annexin V, CF488A
0.16% 1.4%
95.2% 3.2%
12
Figure 4. Representative Data from guava Caspase 8-FAM, Caspase 9-SR Assay. Jurkat cells induced with Gamboic Acid-Amide for 4 hours, show a clear distinction between the live (lower left), apoptotic (lower right), and dead populations (upper) acquired on the guava easyCyte HT system. Simultaneous information on Caspase 8, Caspsase 9 activation during the apoptoitc process can be obtained using the assay.
Figure 3. Example data from FlowCellect Cytochrome C assay. Jurkat cells were treated with 1 µM staurosporine for 4 hours and analyzed with the Cytochrome c assay. Staurosporine treated cells (red) demonstrate a significantly lower level of mitochondrial Cytochrome c as compared to uninduced Jurkat cells (blue).
MitoSense Red Dye, which demonstrates high
fluorescence in cells with intact mitochondrial
potential and a reduction in fluorescence on
loss of mitochondrial potential (Figure 2).
The same assay also demonstrated increased
Annexin V binding of samples on treatment,
and the 7-AAD staining was proportional
to the percentage of cells with increased
membrane permeability upon treatment.
The FlowCellect Cytochrome c assay provides
further information on mitochondrial
proteins and is indicative of commitment
to the intrinsic pathway of apoptotic cell
death. The assay enables the quantitation of
the percentage of cells with altered levels
of mitochondrial Cytochrome c, a critical
protein released from the mitochondria into
the cytosol during apoptosis. This triggers the
formation of the apoptosome and downstream
activation of caspases (Figure 1). Unlike
traditional assays measuring Cytochrome c
release, which have been performed using
Western blots, the FlowCellect Cytochrome c
assay is conducive to plate-based cytometry,
enabling the analysis of a larger number of
samples, using much simplified methods.
Figure 3 shows data from staurosporine
treatment of Jurkat cells followed by
analysis with the Cytochrome c assay. The
data demonstrated that staurosporine
treatment under the test conditions resulted
in 92% of Jurkat cells with reduced levels of
mitochondrial Cytochrome c. Thus, uninduced
Jurkat cells (blue population) showed a higher
level of mitochondrial Cytochrome c compared
to Jurkat cells treated with 1 µM staurosporine
(red population), demonstrating a significant
downward shift in the level of Cytochrome c.
Finally, the assay enabled quantitation of the
percentage of cells committed to the intrinsic
process of apoptosis.
The third set of assays used in these studies are
guava Caspase 8 FAM and Caspase 9 SR assays.
Caspases are intracellular proteases actively
involved in the degradation of a wide range
of cellular components during the apoptotic
cell death. As described above, Cytochrome
c release from the mitochondria results in
the formation of the apoptosome, activation
of Caspase 9 and activation of downstream
caspases as shown in Figure 1. Caspase 8 is
a Caspase that has been largely implicated in
the extrinsic pathway of apoptosis, however
recent literature has also demonstrated that
Caspase 8 can be activated downstream of
Caspase 9 in a feedback amplification loop.
Simultaneous analysis of Caspase 8 and
Caspase 9 can provide valuable mechanistic
information during the screening process. The
guava Caspase 8 and Caspase 9 kits employ a
fluorescently-tagged cell permeable caspase
specific FLICA (Fluorescent Inhibitor of Caspase
Activation) reagent which specifically binds
to active caspase molecules. The Caspase 8
7AAD
(RED
-HLo
g)
Caspase 8 FAM (GRN-HLog)
7AAD
(RED
-HLo
g)
Caspase 9 SR (YLW-HLog)
7AAD
(RED
-HLo
g)
Caspase 8 FAM (GRN-HLog)
7AAD
(RED
-HLo
g)
Caspase 9 SR (YLW-HLog)
Figure 5. Representative heat maps of microtiter-plate-based cytometry data for drug cytotoxicity screening. Jurkat cells were treated with about 160 cytotoxic compounds for 4 hours and 24 hours and evaluated using EMD Millipore’s MitoDamage, Cytochrome c, or Caspase 8&9 Kits followed by analysis on the guava easyCyte 8HT instrument. Percent population data were compared in a heat map format using EMD Millipore’s InCyte Software. InCyte software allowed for the quick identification of hit compounds and comparison of all 6 parameters simultaneously as shown in the pie charts above (A); the degree of cytotoxicity is shown at the bottom of the plates in (B).
4 HOURS
24 HOURS
A
B
7-AAd MitoSense red
Annexin Vcaspase 9
cytochromec
caspase 8
0-20% 20-40% 40-60% 60-80% 80-100%
13
Figure 6: The table above shows a summary of the cytoactive compounds tested that were seen to have any positive effect on the Jurkat cells after induction for either 4 or 24 hours.
cytochrome c caspase 8 caspase 9 MitoSense red Annexin V, cF488A 7-AAd
compounds 4 hrs 24 hrs 4 hrs 24 hrs 4 hrs 24 hrs 4 hrs 24 hrs 4 hrs 24 hrs 4 hrs 24 hrs
Gambogic Acid
Rotenone
Aklavine Hydrochloride
Celastrol
Deguelin (-)
Gambogic Acid Amide
Rutilantinone
2,6-Dimethoxyquinone
Juglone
Trichlormethine
Sanguinariane Sulfate
Mitomycin C
Chlorambucil
Chlorhexidine
Chloroquine Diphosphate
Colchicine
Cytarabine
Emetine
Heachlorophene
Mechlorethamine
Methotrexate (+/-)
Quinachrine Hydrochrolride
Thimerosal
Thioguanine
Vinblastine Sulfate
Warfarin
Azaserine
Phenylmercuric Acetate
Mycophenolic Acid
Ouabain
Cantharidin
Phorbol Myristate Acetate
Etoposide
Azacitidine
Cytochalasin E
Benzyl Isothiocyanate
Floxuridine
Niclosamide
Benzalkonium Chloride
Paclitaxel
Sirolimus
Teniposide
Cytochalasin A
Ancitabine Hydrochloride
Tetrandrine
Picropodophyllin
Griseofulvin
Rubescensin A
Nobiletin
Vincristine Sulfate
Epirubicin Hydrochloride
Camptothecin
0-24.9% 25-49.9% 50-74.9% 75-100%
14
FAM reagent fluoresces in the FITC or green
channel while the Caspase 9 reagent fluoresces
in the SR or yellow channel. The FLICA
reagent is used in addition to the dead cell
stain 7-AAD to allow for distinction between
cell death and caspase activity. An example
of caspase response on treating Jurkat cells
with gambogic acid-amide, an inhibitor of
BCl-xL with known cytotoxic activity is shown
in Figure 4. Four populations of cells were
distinguished with both assays. The results
indicated that gambogic acid-amide induced
the activation of both Caspase 8 and Caspase
9 proteases.
We assessed the cytoactive potential of 160
compounds by applying them to Jurkat cells
and screening with FlowCellect MitoDamage
kit, FlowCellect Cytochrome c kit, guava
Caspase 8 and guava Caspase 9 kits.
Multiparameter analysis, combined with the
heat mapping capability of InCyte software
Figure 7: Representative dose response curves, for multiple mitochondrial health and cell health parameters, obtained on treatment of Jurkat cells with multiple concentrations of Camptothecin (A) for 6 hours and Gambogic Acid (B) for 4 hours are shown above.
A
B
1009080706050403020100
0.01 0.1 1µM of inducer
% o
f cel
ls w
ith re
spon
se
10 100
1009080706050403020100
0.1 1µM of inducer
10 100 1000
MitoSense Red
Annexin
7AAD
Caspase 8
Caspase 9
Cytochrome c
% o
f cel
ls w
ith re
spon
se
enabled us to quickly visualize and identify
the most potent compounds for each measure
of cytotoxcity (Figure 5). Representative heat
maps of plate-based cytometry data show both
similarities and differences in mechanisms
of cytotoxicity among compounds tested on
Jurkat cells. As expected, 24 hour exposure to
compounds elicited greater responses than
4-hour exposure.
After eliminating autofluorescent compounds,
19 of the 160 compounds showed response
at 4 hours while 49 of the 160 showed
responses at 24 hours (Figure 6). Compounds
such as hexachlorophene showed a change
in MitoSense Red (measures mitochondrial
potential) but no cytochrome c release.
Other compounds such as juglone showed
a change in cytochrome c at 4 hours and a
lower response in MitoSense until 24 hours.
Several compounds showed greater impacts
on cytochrome c release and mitochondrial
potential changes than other parameters,
while others showed impact on all parameters
in parallel. Following this initial screening, a
subset of compounds was taken for further
dose response analysis.
The assays described can also be utilized
to obtain dose response information on
specific compounds of interest. Thus, results
demonstrate that camptothecin, an inhibitor
of DNA topoisomerase I caused almost parallel
changes in mitochondrial membrane potential,
annexin V binding, and cytochrome c release,
indicating apoptosis through the intrinsic
pathway, with no cell death observed under
conditions tested. Caspase 8 and caspase 9
activity were both observed on treatment with
camptothecin, which agrees with currently
accepted models of its mode of action.
Results obtained with gambogic acid
demonstrate that loss of mitochondrial
membrane potential occurs at significantly
lower concentrations when compared to the
other apoptotic responses. The percentage of
cells demonstrating changes in cytochrome
c release, annexin V detection and caspase
8 and 9 activity were less than those seen
with MitoSense Red for several of the same
treatment conditions. Cytotoxic activity, as
detected by the increased percentage of cells
that are 7-AAD positive, was observed at much
higher concentrations when compared to the
other assays. The detection of both caspase 8
and 9 activities on gambogic acid treatment
is consistent with data in the literature. It’s
interesting to note that a positive but reduced
level of caspase 8 activity was detected
when compared to capsase 9. These dose
response curves suggest that gambogic acid,
cytochrome c-mediated caspase 9 activation
results in downstream activation of caspase 8.
Investigating mitochondrial depolarization and
cytochrome c release in addition to caspases
and annexin V, thus lead to crucial information
in understanding the mechanism of compound
action.
15
conclusionsEMD Millipore’s FlowCellect MitoDamage
and Cytochrome c kits along with Caspase
8-FAM & Caspase 9-SR kits, in combination
with the guava easyCyte HT system and
InCyte software, allowed for rapid and facile
plate-based screening of 160 cytoactive
compounds simultaneously and enabled quick
identification of hit compounds for each
effect. Use of MitoSense Red and Cytochrome
c, in particular, provided detection of early
changes within the mitochondria that preceded
complete apoptosis and cell death.
Dose response curves generated provided
further information on sequence of impacts
and mechanism of action. Results were
obtained from dose response analysis of
camptothecin and gambogic acid, both of
which exhibit caspase 8 and caspase 9 activity.
The downstream detection of caspase 8 with
gambogic acid following caspase-9 activation
suggests its involvement in an apoptosis
amplification loop.
Correlating mitochondrial and cell health
markers provides for a deeper understanding
of whether the impacts are upstream or
downstream of mitochondria and whether
they are are induced through receptor-
mediated or mitochondrial pathways of death.
These conclusions support that evaluation of
multiple parameters is of great importance in
understanding the mechanism of action of
cytoactive compounds.
REFERENCES1. Zamzami N, Kroemer G. The mitochondrion in apoptosis:
how Pandora’s box opens. Nat Rev Mol Cell Biol. 2001;2:67-71
2. Jiang X, Wang X. Cytochrome c-mediated apoptosis. Annu Rev Biochem 2004;73:87–106.
3. Wagner BK, Kitami T, Gilbert TJ, Peck D, Ramanathan A, Schreiber SL, Golub TR, Mootha VK. Large-scale chemical dissection of mitochondrial function. Nat Biotechnol. 2008 Mar;26(3):343-51, 2008 Jul;26(7):831
4. Clor, J. et al. Rapid Cell-Based Assays Using Multiplexed Mitochondrial and Cell Health Markers. Cellutions 2010:Vol. 2, p. 16-18.
RELATED PRODUCTS
description catalogue No.
FlowCellect MitoDamage Kit FCCH100106
FlowCellect Cytochrome C Kit FCCH100110
guava Caspase 8-FAM & Caspase 9-SR Kit 4500-0640
Available from www.millipore.com.
Well of Tissue Culture Plate
Feet = “Classic” Millicell insert“Wings” = Hanging Millicell Insert
Cell Culture Insert
Medium
Cells
Wing
Concentration gradient
Microporous Membrane
Wing
Upper well with cells
Membrane
Lower well with chemoattractant and/or drug
Feet
Suspension cells Adherent cells
A
B
16
AbstractCell migration occurs in a variety of biological
processes including wound healing, embryonic
development, and immune responses.
Existing chemotaxis assays used to assess
cell migration employ the Boyden chamber,
which lacks real-time imaging capability and
does not maintain well-defined biomolecular
gradients. To overcome these limitations, a cell
migration assay was developed which enables
real-time monitoring of single cells on a slide-
like platform that maintains a stable linear
gradient.
IntroductionCell migration is stimulated and directed by
interaction of cells with the extracellular matrix
(ECM), neighboring cells, or chemoattractants.
During embryogenesis, cell migration
participates in nearly all morphogenic
processes ranging from gastrulation to neural
development. In the adult organism, cell
migration contributes to physiological and
pathological conditions, and is central to
development of therapeutics affecting wound
healing and tumor metastasis. Specifically,
inhibiting tumor invasion by blocking tumor
cell chemotaxis has been a major focus of
research.
The most widely accepted cell migration
assay is the Boyden chamber assay, using
a two-chamber multiwell plate in which a
membrane in each well provides a porous
interface between two chambers (Figure 1A).
Chemoattract is placed in the lower chamber,
and the system is allowed to equilibrate, with
the expectation that a gradient would form
between the upper and lower wells. However, in
reality, very steep gradients can form along a
single axis perpendicular to the surface of the
membrane, resulting in a lower-than-expected
difference in chemoattractant concentration
between upper and lower wells. (Figure
1B). As a result, this method is unsuitable
for correlating specific cell responses with
particular gradient characteristics (i.e., slope,
concentration, temporal evolution, etc),
preventing its use for studying multi-gradient
signal integration.
New Chemotaxis Assay for Single Cell Analysis Using a Microscale Migration Chipyi (Joy) Zhou, cristina Moore, christine chen, Vi chu
EMD Millipore
Figure 1: (A) Principle of Boyden Chamber Assay. (B) Change in chemoattractant concentration with respect to equilibration time in a 24-well Millicell plate (PET, 8 µm). The concentration in the bottom compartment was defined as ‘1’ and the original relative concentration in the upper compartment was 0.05. After 8 hours, the relative concentration in the upper compartment was around 0.1.
0.200.180.160.140.120.100.080.060.040.02
00 1 2 3 4 5 6 7 8
Time (hr)
8 µm upper compartmentOriginal upper compartment
Rela
tive
Conc
entr
atio
n
A
B
17
70 µm1 mm1 mm
8 hrs
24 hrs
48 hrs
Figure 2. Diagram and corresponding cross section of the Millicell µ-migration assay slide.
Figure 4. Photomicrograph of migrated cells with colored lines following paths.
Figure 3. (A) Side view of a µ-migration chamber and representative photomicrographs of the slit showing linear fluorescence gradient across the slit. (B) Normalized fluorescence intensity across the slit with respect to distance and time.
In contrast, the Millicell® µ-Migration Slide
features a linear concentration gradient stable
for ≥ 48 hours, which provides reproducibility
and the capability to accurately compare
effects of chemoattractants on mechanisms of
migration. The platform also helps distinguish
chemotaxis from random movement, and
multiparametric analysis enables even greater
mechanistic insight.
MethodsThe Millicell µ-migration assay slide consists
of two nearly infinite-volume reservoirs
connected by a thin (70 µm deep and 1 mm
wide) observation slit (Figure 2). A linear
gradient is defined, reaching a steady state
upon diffusion. This assay is compatible with
real-time microscopy imaging for long-term
chemotactical observation.
We analyzed the migration of HT1080,
HUVECs, NIH 3T3 and MDA-MB 231 cells on a
collagen-coated surface in response to varying
concentrations of fetal calf serum (FCS). For
each cell line, at least 30 cells were tracked
using a manual tracking plug-in software.
Using this software, we monitored directed
distance migrated, speed of directed migration,
and numbers of cells moving towards or away
from the source.
resultsTo assess stability and linearity of the
gradient, we applied a fluorescent compound
in compartment labeled “6” and measured
fluorescence across the slit with respect to
time (Figure 3A). Relative fluorescence intensity
was plotted with respect to distance across
the slit (Figure 3B) at varying time points.
normalized intensity
t = 2 hrs
t = 4 hrs
t = 8 hrs
t = 24 hrs
t = 48 hrs
Linear (t = 2 hrs)
Linear (t = 4 hrs)
Linear (t = 8 hrs)
Linear (t = 24 hrs)
Linear (t = 48 hrs)
norm
aliz
ed in
tens
ity
0.45
0.2 0.4 0.6 0.8 1.0
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05distance (mm)
normalized intensity
t = 2 hrs
t = 4 hrs
t = 8 hrs
t = 24 hrs
t = 48 hrs
Linear (t = 2 hrs)
Linear (t = 4 hrs)
Linear (t = 8 hrs)
Linear (t = 24 hrs)
Linear (t = 48 hrs)
norm
aliz
ed in
tens
ity
0.45
0.2 0.4 0.6 0.8 1.0
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05distance (mm)
normalized intensity
t = 2 hrs
t = 4 hrs
t = 8 hrs
t = 24 hrs
t = 48 hrs
Linear (t = 2 hrs)
Linear (t = 4 hrs)
Linear (t = 8 hrs)
Linear (t = 24 hrs)
Linear (t = 48 hrs)
norm
aliz
ed in
tens
ity
0.45
0.2 0.4 0.6 0.8 1.0
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05distance (mm)
normalized intensity
t = 2 hrs
t = 4 hrs
t = 8 hrs
t = 24 hrs
t = 48 hrs
Linear (t = 2 hrs)
Linear (t = 4 hrs)
Linear (t = 8 hrs)
Linear (t = 24 hrs)
Linear (t = 48 hrs)
norm
aliz
ed in
tens
ity
0.45
0.2 0.4 0.6 0.8 1.0
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05distance (mm)
normalized intensity
t = 2 hrs
t = 4 hrs
t = 8 hrs
t = 24 hrs
t = 48 hrs
Linear (t = 2 hrs)
Linear (t = 4 hrs)
Linear (t = 8 hrs)
Linear (t = 24 hrs)
Linear (t = 48 hrs)
norm
aliz
ed in
tens
ity
0.45
0.2 0.4 0.6 0.8 1.0
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05distance (mm)
••
••
70 µm1 mm
CrossSection*
ObjectiveLens
1
12
3
66
7
7
8 8
2 3
*Cross Section indicating the place of cells, chemoattractant, and gradient
A
B c
A B
18
RELATED PRODUCTS
description qty catalogue No.
NEW Millicell µ-Migration Assay Kit 1 kit(4 slides,
12 assays)MMA205
required Equipment/reagentsAccutase™ Cell Dissociation Solution 100 mL SCR005
Dulbecco’s Phosphate Buffered Saline, 1X ES Cell Qualified 500 mL BSS-1005-B
EmbryoMax® ES Cell Qualified Ultra Pure Water, sterile H2O 500 mL TMS-006-B
EmbryoMax ES Cell Qualified Ultra Pure Water, sterile H2O 100 mL TMS-006-C
Scepter™ Handheld Automated Cell Counter 1 ea PHCC00000
Figure 5: A typical data set to assess the effects of serum concentrations (0, 5, 10, and 15%) on the migration propensity of HT1080 cell line on collagen-coated surface. (A) Plot graphs of tracked cells (B) Migration index of tracked cells (C) Migration velocities of tracked cells towards different concentration of chemoattrant (FCS).
Figure 6: Four different cell lines are used to assess this assay, which respond to a neutral and a chemoattrant respectively. (A) Percentage of cell population moving towards the chemoattrant. (B) Migration index of tracked cells moving towards the chemoattrant.
We found that the maximum concentration
reached 33% of the applied concentration,
which was due to the dilution and diffusion.
Furthermore, the gradient was very linear and
was stable for over 48 hours.
Migrating cells were visualized in real time
using microscopy, and Figure 4 shows a typical
example of cells with tracking lines showing
migratory paths. Enhanced optical imaging
makes the µ-migration slide especially suitable
for high-quality and fluorescence microscopy,
perfect for observing single cells.
Plotting the migration paths of tracked HT1080
cells (Figure 5A) showed that cells migrated
directionally in response to 10% and 15% FCS
(Figure 5B), indicated by increased positive
y-axis migration. Migration velocity reached a
maximum in response to 10% FCS (Figure 5C).
Migration of four different cell lines to varying
concentrations of FCS was measured using
the µ-migration slide (Figure 6). The highest
percentage of migratory HUVEC cells was
obtained using 2% serum whereas the other
three cell lines exhibited increased % migration
(A) as well as increased directional migration
(B) with increasing serum concentration.
conclusionsThese studies using the Millicell µ-migration
assay reveal advantages such as the capability
for performing chemotactical observations
over long time periods, linear and stable
concentration gradients, and the possibility of
high-quality microscopy for observing single
cells with high resolution.
This assay promises to be a useful tool for
monitoring the effects of chemoattractants
on adherent cell lines. Our results provide a
reference point from which to build upon
future studies aimed at comparing the effects
of signaling molecules and growth factors
on the migration propensities of cells in
tumors, wounds, developing tissues, immune
responses, and other biological systems defined
by active cell migration.
0% 0%
5%
ECS
ECS
0% 0%
10% 15%
ECS
ECS
Mig
ratio
n In
dex
0% 5% 10% 15%
0.25
0.2
0.15
0.1
0.05
-0.05
-0.1
0
Concentration of FCS
x-forward migration indexy-forward migration index
Mki
grat
ion
Velo
city
(µm
/min
)
0% 5% 10% 15%
0.140.120.1
0.080.060.040.02
-0.02-0.04-0.06
0
Concentration of FCS
x-forward migration velocityy-forward migration velocity
0% 2%HUVEC
0% 10%HT1080
0% 10%NIH3T3
0% 10%MDA-
MB-231
85%80%75%70%
60%65%
55%50%45%
0% 2%
HUVEC
0% 10%
HT1080
0% 10%
NIH3T3
0% 10%
MDA-MB-231
0.40.30.2
00.1
-0.1-0.2-0.3-0.4-0.5
x-forward migration indexy-forward migration index
Mig
ratio
n In
dex
0% 5% 10% 15%
0.25
0.2
0.15
0.1
0.05
-0.05
-0.1
0
Concentration of FCS
x-forward migration indexy-forward migration index
Mki
grat
ion
Velo
city
(µm
/min
)
0% 5% 10% 15%
0.140.120.1
0.080.060.040.02
-0.02-0.04-0.06
0
Concentration of FCS
x-forward migration velocityy-forward migration velocity
0% 2%HUVEC
0% 10%HT1080
0% 10%NIH3T3
0% 10%MDA-
MB-231
85%80%75%70%
60%65%
55%50%45%
0% 2%
HUVEC
0% 10%
HT1080
0% 10%
NIH3T3
0% 10%
MDA-MB-231
0.40.30.2
00.1
-0.1-0.2-0.3-0.4-0.5
x-forward migration indexy-forward migration index
Avaliable from www.millipore.com/umigration.
19
IntroductionThe Scepter cell counter combines the ease of
automated instrumentation and the accuracy
of Coulter impedance-based particle detection
in an affordable, handheld format. The
instrument, which is the size of a pipette, uses
a combination of analog and digital hardware
for sensing, signal processing, data storage,
and graphical display. The precision-made,
consumable polymer sensor has a laser-drilled
aperture in its cell-sensing zone that enables
the instrument to use the Coulter principle to
discriminate cell size and cell volume at sub-
micron and sub-picoliter resolution.
The corollary to this principle is that the size
of the sensor’s aperture defines the diameter
range of cells that can be counted accurately
using the Scepter cell counter. In its initial
version, the Scepter cell counter included one
sensor with a 60 µm aperture. The experiments
described here showed that, by adding the
option of using a sensor with a smaller
aperture (40 µm), the Scepter cell counter was
able to accurately and precisely count a much
broader range of cell types, including small
cells (less than 6 µm in diameter); like stem
cells, neurons, and PBMCs. Because the Scepter
cell counter measures volume using the
Coulter Principle, it can properly discriminate
cells from debris and background unlike
vision-based techniques, which rely on object
recognition software and cannot accurately
detect small cells. We also show that the cell
counter was able to count samples with greatly
increased cell concentration using the 40 µm
sensor.
Materials and Methodscell types testedWe have previously described the cell lines
validated with the 60 µm sensor1. For this
study, we focused on human red blood cells
(US Biologicals, R1300-25), peripheral blood
mononuclear cells (PBMCs, Lonza, CC-2702),
peripheral blood neutrophils (Allcells, PB016),
CD3+ T cells (Astarte Biologics, 1017-253OC10),
B cell lymphocytes (Astarte Biologics, 7243703),
monocytes (Lampire Biological, 7243703), rat
whole blood (Lampire Biological, 7204309),
mouse whole blood (Lampire Biological,
7207009), Saccharomyces cerevisiae (ATCC),
Pichia pastoris (ATCC), Jurkat cells (ATCC), NIH
3T3 cells (ATCC), U266 cells (ATCC), ENStem-A
neural progenitors (EMD Millipore, SCC003),
ReNcell immortalized human neural progenitors
(EMD Millipore, SCC008), rat dorsal root ganglia
(Lonza, R-DRG-505), and rat hippocampal
astrocytes (R-HiAs-521).
Sample preparationSingle-cell suspensions were diluted with
phosphate-buffered saline (1X EmbryoMax
PBS, EMD Millipore) for sufficient conductivity
and counted using a Z2™ Coulter Counter®
(Beckman Coulter). Dilution series were
prepared in 1.5 mL microcentrifuge tubes. The
dilutions ranged from 50,000 to 1,500,000
cells/mL with a minimum sample volume of
100 µL. The starting cell concentration was
divided by the fold dilution at each serial
dilution step to determine theoretical cell
concentrations. Four replicates of each dilution
were prepared for Scepter 2.0 counting.
The New Scepter 2.0 Cell CounterEnables the Analysis of a Wider Range of Cell Sizes and Types With High Precision
Janet Smith, Melinda Wilson, Kathleen ongena
EMD Millipore
20
Scepter cell countingThe Scepter cell counter was used to count cell
samples by following the detailed on-screen
instructions for each step of the counting
process. Briefly, the user depresses the plunger,
submerges the sensor into the solution,
then releases the plunger to draw 50 µL or
75 µL of cell suspension into the sensor. The
Scepter cell counter detects each cell passing
through the sensor’s aperture, calculates the
cell concentration, and displays a histogram
of cell size as a function of cell volume or cell
diameter on its screen.
cell counting by other methodsCounts of each cell line were also performed
using the Coulter Counter, an automated
vision-based counter such as a Vi-CELL®
(Beckman Coulter), TC10™ (Bio-Rad), or
Countess® (Life Technologies) system, and
a hemocytometer. Counts were performed
according to manufacturer’s instructions using
the same cell starting suspension and identical
dilutions.
data analysisHistograms were gated manually, using the
same upper and lower limits as used for the
Coulter Counter, to exclude cell debris from cell
concentration calculations. Histograms were
uploaded to a personal computer using the
the new downloadable Scepter Software Pro
and USB cable. Meta-analyses were conducted
by exporting the data to Microsoft Excel®
Software.
resultsThe cell types tested with the Scepter cell
counter, either with the 60 µm sensor (reported
previously) or with the 40 µm sensor, yielded
interpretable histograms that could be gated
and used to calculate cell concentration, mean
cell size and cell diameter. Examples of these
histograms are shown in Figure 1.
A
c
E
B
d
F
Figure 1. The Scepter cell counter is compatible with a wide range of cell types, including:a) Pichia pastoris yeastb) ReNcell neural progenitor cellsc) Neutrophilsd) Monocytese) T cells (CD3+)f) B cells (human)
21
A B
Figure 2. NIH 3T3 cells (13.5 µm in diameter) were more accurately counted using the 60 µm sensor (B) than by using the 40 µm sensor (A). Lower gates for both histograms were set to 7.6 µm.
To determine whether 60 µm or 40 µm
aperture sensors should be used for certain
cell types, we counted these cell types with
both sensor types. For example, for cells larger
than 13 µm in diameter, the 40 µm sensor was
not able to accurately count cells larger than
18 µm (in the right tail of the histogram peak,
Figure 2A), resulting in underestimations of
cell concentration. For these cells, the 60 µm
sensor provided greater accuracy and precision
(data not shown). Cells smaller than 7 µm, such
as peripheral blood mononuclear cells (PBMCs),
were best counted with the 40 µm sensor.
Comparing average %CVs across all replicates
and dilutions (Figure 3), we found that Scepter
2.0 counting was more precise than automated
vision-based counting and hemocytometry
when counting these small cells.
Cell types that were between 6 and 14 µm in
diameter, such as Jurkat cells (used in Figure
4) were successfully counted with both 40
µm and 60 µm sensors. We therefore used
Jurkat cells to validate reproducibility of cell
analyses between Scepter devices and between
sensor types. The overlapping data points
and small error bars in Figure 4 show that,
regardless of which of three Scepter devices
were used, and regardless of which sensor
was used, cell concentration measurements
are precise, accurate, and reliable, even at cell
concentrations as high as 1,500,000 cells/mL.
Figure 4. Calculated concentrations of Jurkat cells using Scepter 2.0 counting are highly precise, regardless of device used, sensor aperture size, or sample dilution.
353025
Aver
age
(%CV
)
Scepter Counter(40 µm sensor)
Automated Vision-based Counter
Hemocytometer Z2 Coulter Counter
Counting Method
20151050
20
15
% C
oeffi
cien
t of
Var
iatio
n (%
CV)
Scepter Counter(40 µm sensor)
Average %CVZ2 Coulter Counter
RBCs
PBMCs
Automated Vision-based Counter
Hemocytometer
Counting Method
20
5
0
150
100
125
75
50
25
00 25 50 75 100 125 150
Theoretical Concentration (cells mL x 10,000)
Scepter #1: 40 µmScepter #2: 40 µmScepter #3: 40 µmScepter #4: 60 µm
Avg
Conc
entr
atio
n(c
ells
mL
x 10
,000
)
Figure 3. The Scepter 2.0 cell counter counts PBMCs with greater precision than other counting methods, as reflected by low average coefficient of variation.
22
description quantity catalog No.
Scepter 2.0 Handheld Automated cell counter
with 40 µm Scepter Sensors (50 Pack) 1 PHCC20040
with 60 µm Scepter Sensors (50 Pack) 1 PHCC20060
Includes:
Scepter Cell Counter 1
Downloadable Scepter Software 1
O-Rings 2
Scepter Test Beads 1 PHCCBEADS
Scepter USB Cable 1 PHCCCABLE
Scepter Sensors, 60 µm 50 PHCC60050
500 PHCC60500
Scepter Sensors, 40 µm 50 PHCC40050
500 PHCC40500
Universal Power Adapter 1 PHCCP0WER
Scepter O-Ring Kit, includes 2 O-rings and 1 filter cover 1 PHCC0CLIP
RELATED PRODUCTS
To compare the overall precision of Scepter
2.0 counting with a 40 µm sensor, the percent
coefficient of variation (%CV) was calculated
for each dilution of each cell line for each
counting system, and the average %CV across
12 cell types was recorded as a measure of
precision. Scepter 2.0 counting displayed
smaller average %CVs compared to automated
vision-based counting and hemocytometry
across all tested cell lines (Figure 5).
The overall relative accuracy of Scepter
2.0 counting using the 40 µm sensor was
evaluated by comparing cell counts obtained
with the Scepter cell counter with counts
obtained with the Z2 Coulter Counter (Figure
6). All the points fall on or close to the line of
perfect agreement between the two methods,
indicating that Scepter counting is a viable
alternative to laboratories currently relying
on large, benchtop automated counters for
determining cell concentration.
conclusionsToday’s cell biology laboratories routinely use
multiple cell types within a given laboratory.
Often, even an individual researcher routinely
uses a variety of cell types with diverse
characteristics to address particular research
questions. For these cases, a personal cell
counting device that still retains superior
versatility and precision can improve cell-based
assays and accelerate workflows. Extending
the capabilities of the Scepter cell counter to
include cellular blood components, neural cells,
and yeast, in particular, has the potential to
advance immunology, neurodegeneration, and
systems biology research, some of the fastest
growing fields of study in life science.
REFERENCES1. Ongena, K et al. The Scepter Cell Counter Performs With
High Precision and Speed Across Multiple Cell Lines. Cellutions 2010. Vol. 1: p. 5-7.
Figure 6. Plotting log of Coulter counts vs. log of Scepter counts shows that, across all cell lines tested with the 40 µm sensor, Scepter counts match Coulter counts.
Figure 5. Average %CV with respect to concentration of the Scepter cell counter with 40 µm sensor and other cell counting methods.
45%40%35%30%25%20%15%10%5%
00 25 50 75 100 125 150
Theoretical Concentration (cells mL x 10,000)
Z2 Coulter CounterHemocytometerVision-based Automated CounterScepter Counter with 40 µm Sensors
% C
V
1,000,000
100,000
10,000
1,0001,000 10,000 100,000 1,000,000
Cell Concentration (cells/mL, Scepter counter)
Cell
Conc
entr
atio
n (c
ells
/mL,
Cou
lter C
ount
er)
SCATTERPLOT OF CONC. COULTER Z2 VS. CONC. SCEPTER 40 µM SENSORS
Available from www.millipore.com/scepter.
23
Scepter 2.0 is Here! The Next Generation of Automated Cell Counting
For the latest application data, see the article on page xx of this issue!
prodUct HIGHLIGHt
Integrated display
• Histogram data on cell populations
• Cell concentration, mean cell volume,
and cell size
• Can apply custom gating
• Gain insight into cell health
plastic consumable Sensors • Detect cells of varying size ranges
with 2 sensor types
• Discriminate cell sizes with
sub-micron resolution
• Discriminate cell volumes with
sub-picoliter resolution
Handheld pipette
• Compact, easy to use
• Ergonomic action feels like pipetting
• On-screen instructions
Scepter Features
• count more cell types
• Expanded dynamic ranges
• Intuitive software platform
• Easier than ever to get the right answer fast
NEW Scepter Software pro• compare several samples and data sets side by side using histogram overlay and
multiparametric tables
• Save and create gating methods to be used from one experiment to the next
• create attractive graphical presentations and reports with your data
Guava, Millicell, and EmbryoMax are registered trademarks and easyCyte, FlowCellect, InCyte, Accutase, and Scepter are trademarks of Millipore Corporation.InhibitorSelect and the M logo are trademarks of Merck KGaA, Darmstadt, Germany.Z2 is a trademark and Coulter Counter and VI-CELL are registered trademarks of Beckman Coulter, Inc. Countess is a registered trademark of Molecular Probes, Inc. TC10 is a trademark of Bio-Rad Laboratories, Inc. IncuCyte is a registered trademark of Essen Instruments, Inc.ReNcell is a registered trademark of ReNeuron Ltd. Excel is a registered trademark of Microsoft Corporation.Lit No. PR1435EN00 LS-SBU-11-04126 Printed in the USA 3/2011 © 2011 Millipore Corporation. All rights reserved.
CONTACT USIn the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)
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