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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

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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

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• Up to six-color detection

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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

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• Wash vial offers a

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+ MORE PARAMETERS

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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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