GE Healthcare
Cell integrity assays
High-content analysis of essential cell integrity and
toxicity parameters using the IN Cell Analysis System
In recent years the development of high-content analysis
has allowed the development of a number of cellular assays
that have the potential to provide information on potential
drug toxicity earlier in the discovery process.
Critical cell functions that can be analyzed using automated
and manual microscopes with GE Healthcare’s cellular
reagent technologies and image analysis software in
drug toxicity testing include:
Cellular toxicity can occur through a diverse range of
mechanisms that disrupt cellular integrity. Membranesoluble or pore-forming compounds may act directly on the
cytoplasmic membrane and prevent the cell maintaining
homeostatic integrity, leading to necrosis. Other compounds
may act indirectly to disrupt the cell’s biochemical,
synthetic, or signaling integrity, leading to apoptosis. Further
compounds may act directly or indirectly to damage the
cell’s genetic integrity, resulting in inheritable mutation,
disruption of proliferative integrity, or apoptosis.
• Membrane integrity
• Proliferative integrity
• Organelle integrity
• Nuclear integrity
• Genetic integrity
• Intracellular signaling integrity
Membrane integrity
Cell toxicity and death caused by drugs can occur through
necrosis or apoptosis. In some cases these events may
occur sequentially or in parallel depending on the dose and
duration of exposure of cells to a test compound. There are
several morphological and biochemical differences between
necrosis and apoptosis and these may be detected using
high-content analysis (HCA) markers (Table 1).
Necrosis typically occurs when cells are exposed to an injury
that damages the plasma membrane and prevents the cell
from maintaining homeostasis. Necrosis can be readily
detected by imaging the uptake of cell-impermeable
fluorescent dyes such as propidium iodide into cells with
damaged plasma membranes (Fig 1).
In contrast to passive necrosis, apoptosis is an active energy
requiring process that occurs under normal physiological
conditions where cells are triggered to self-destruct.
Apoptotic cells show characteristic morphological and
biochemical features including nuclear and cytoplasmic
condensation, membrane blebbing, and membrane inversion.
In the early stages of apoptosis the anionic lipid
phosphatidylserine (PS) translocates from the inner side of
the plasma membrane to the outer layer. This inversion can
be imaged using fluorescently labeled annexin-V (a calciumdependent phospholipid-binding protein) as a marker for
early apoptosis (Fig 2).
Analysis of cell morphology is a powerful and informative
adjunct to the use of fluorescent dyes for investigating toxic
action of candidate drugs. Analysis using cytoplasmic and
nuclear shape descriptors (Fig 3A) allows rapid quantitation
of cells exhibiting normal and aberrant morphology (Fig 3B)
as a measure of drug effects on cellular integrity.
Table 1. Characteristics and HCA markers for necrosis and apoptosis.
Characteristics of necrosis
Characteristics of apoptosis
Loss of plasma membrane integrity
Membrane blebbing
Swelling of cytoplasm
Shrinkage of cytoplasm and nucleus
Loss of homeostasis
Alterations in membrane symmetry
Total cell lysis and dissolution of contents
Cell fragmentation into smaller bodies
HCA markers for necrosis
HCA markers for apoptosis
Propidium iodide uptake
Annexin V binding
Increase in cell area
Decrease in nuclear area
Decrease in cell number
Chromatin condensation
Nuclear fragmentation and Increase in sub-nuclear objects
Fig 1A. Intact (blue)
and necrotic (red)
cells identified by
staining with
Hoechst and
propidium iodide
respectively. Image
acquired on IN Cell
Analyzer 1000.
Fig 1B. Induction
of cellular necrosis
measured by uptake
of propidium iodide
following treatment
with increasing
concentrations of
test compounds
for 6 h.
Fig 2A. U2OS cells
treated with 20-μM
staurosporine for 24 h
then stained with
10-μM Hoechst, 10-μM
propidium iodide, and
500-ng/ml FITClabeled annexin V.
Image acquired on IN
Cell Analyzer 1000.
Hoechst = blue,
propidium iodide =
red, and annexin V =
green.
Fig 2B. Binding of
FITC-labeled annexin
V to HeLa cells
incubated with
increasing
concentrations of
ionomycin for 4 h.
Fig 3A. IN Cell
Analyzer 1000
cellular morphology
analysis. Cells
showing normal and
apoptotic (arrow)
morphology were
categorized by
selecting individual
cells with
representative
morphology (inset).
Fig 3B. Automated
classification of cell
morphology. Four
automatically
selected parameters
(selected features,
top) were sufficient
to separate the cells
in Figure 3A into two
distinct populations
(scatterplot, bottom).
The selected
apoptotic cell (arrow)
is the same as that
identified in Fig 3A.
Proliferative integrity
Fig 4. HeLa cells
incubated (A) In the
absence of colchicine
(B) In the presence of
colchicine. Cells were
fixed in ethanol,
stained with
propidium iodide, and
imaged on IN Cell
Analyzer 1000 for
measurement of
DNA content.
The cell cycle is of key importance to many areas of drug
discovery. This fundamental process provides on the one
hand the opportunity to discover new targets for anticancer
agents and improved chemotherapeutics, and on the other
hand requires the testing of drugs and targets in other
therapeutic areas for undesirable effects on the cell cycle.
Measurement of DNA content by flow cytometry of fixed
cells stained with fluorescent dyes such as propidium iodide
is a standard method of analyzing cell cycle distribution.
Performing the same analysis using high-throughput imaging
(Figs 4 and 5) provides a significant increase in throughput
coupled with the ability to multiplex cell cycle analysis
determined by DNA content with other parameters.
Conventional immmunodetection procedures for detecting
5-bromo-2’-deoxyuridine (BrdU) incorporation into the DNA of
replicating cells use acid or alkali denaturation to allow
access of the anti-BrdU antibody. However, these methods
can significantly alter cell morphology and preclude the use
of additional cellular probes.
To enable the use of BrdU assays for HCA, nuclease
treatment is applied during incubation with monoclonal antiBrdU to allow antibody access without adversely affecting
cell morphology or compromising the signal from multiplexed
fluorescent probes.
Fig 5. DNA content
histogram analysis
of HeLa cells shown
in Figures 4A and 4B.
Cells incubated in
the presence of
colchicine show
significant
accumulation of cells
in G2 and M.
Detection with a Cy™5-labeled second antibody allows BrdU
incorporation to be multiplexed with GFP (Fig 6A) or analyzed
with other cellular markers.
For further in-depth analysis of the effects of compounds on
cell cycle and proliferation, GE Healthcare has developed two
dynamic cell cycle sensors based on EGFP fusion proteins
(Fig 7). Coupled with automated image analysis modules,
these G2/M and G1/S Cell Cycle Phase Markers (CCPMs) allow
detailed cell-by-cell analysis for effects of candidate drugs on
cell cycle checkpoint progression, delay, and arrest. Imaging
of CCPMs can be multiplexed with imaging of DNA content
and BrdU incorporation (Fig 6A) to yield a highly informative
picture of drug effects on the cell cycle (Fig 8).
Fig 6A. BrdU
incorporation
detected with the
Cell Proliferation
Fluorescence Assay
in G2/M Cell Cycle
Phase Marker
expressing U2OS
cells. Image acquired
on IN Cell Analyzer
1000 (DNA, GFP,
BrdU).
Fig 6B. BrdU
incorporation
measured in U2OS
cells in the presence
of increasing
concentrations of
mitomycin C.
Fig 7. The G2/M CCPM (top) follows the expression and degradation of
cyclin B1 as a marker for cells transitioning from G2 to M. G2 cells
(arrow) express the CCPM in the cytoplasm with the fusion protein
undergoing translocation to the nucleus in prophase and reaching
maximal intensity at mitosis. Destruction of the sensor post mitosis
(under control of the cyclin B1 D-box) resets the sensor in G1 daughter
cells (arrows) ready for a further cycle. The G1/S CCPM (bottom) follows
the subcellular location of DNA helicase B. Expression in M phase cells
is uniform (arrow) but segregates rapidly to nuclei in G1 daughter
cells (arrows) with export to the cytoplasm as cells transition through
S phase into G2 where the sensor is restricted to the cytoplasm.
Fig 8. Multiplexed analysis of DNA content, G1/S transition and BrdU
incorporation. U2OS cells expressing the G1/S CCPM were incubated in
the presence and absence of a test compound and pulsed with BrdU
for 1 h prior to imaging on IN Cell Analyzer 1000. DNA content was
measured by staining with Hoechst nuclear dye and BrdU
incorporation measured with the Cell Proliferation Fluorescence Assay.
Each sphere represents data from a single cell, with CCPM data
represented by the size of each sphere. In this assay treatment with
compound A induced an increase in DNA content from 2n/4n to 4n/8n
with associated mitotic by-pass resulting in a significant proportion of
8n cells in G1 (large red spheres at 8nDNA).
Organelle integrity
Changes in the shape, distribution, or other characteristics of
subcellular organelles can be an important indicator of toxicity
in cellular assays. For example, swelling of mitochondria
accompanies homeostatic disruption in the early stages of cell
necrosis, and leakage of proteins and other factors from
mitochondria is an early indicator of apoptosis.
Use of the IN Cell Developer Toolbox (see page 15) allows
powerful procedures to be constructed for HCA using
fluorescent dye and protein organelle markers to detect
changes in fluorescence intensity, distribution, and
morphology accompanying toxicity (Fig 9).
Fig 9A. U2OS cells transiently expressing an Emerald-FP
fusion protein targeted to mitochondria. Image acquired on
IN Cell Analyzer 1000. Hoechst = blue, Emerald FP = green,
and Mitotracker™ Red = red.
Fig 9B. Analysis of fusion protein expression and retention
in mitochondria using IN Cell Developer Toolbox.
Nuclear integrity
Changes in the number, size, and shape of nuclei in HCA
images are a simple but powerful indicator of toxic effects in
cells exposed to test compounds. Decreases in nuclear
number/image may indicate inhibitory effects on the cell cycle
or may be due to loss of cells through lysis depending on the
duration of exposure. Similarly, changes in nuclear size
(Fig 10) may be indicative of cell cycle blockage in G2 (increase
in nuclear size) or apoptotic cell death (decrease in nuclear size
with chromatin condensation).
In the advanced stages of apoptosis many nuclei will show
clear breakdown into two or more fragments (Fig 10). These
parameters can readily be quantitated by HCA (Fig 11) using a
range of IN Cell Analyzer Image Analysis Modules and can be
applied to any assay using a nuclear stain to gain valuable
additional information on compound toxicity.
Fig 10. Nuclear changes associated with drug
Fig 11. Analysis of nuclear size and
toxicity. Taxol-treated cells (bottom) show
significant changes in nuclear morphology
compared with control cells (top) including
fragmentation (a) and swelling (b) as well as a
significant decrease in numbers of
nuclei/image. Images acquired on IN Cell
Analyzer 1000.
fragmentation in taxol-treated cells using IN Cell
Analyzer Image Analysis Modules.
Genetic integrity
Micronucleus induction is a key characteristic of genotoxic
compounds. Analysis of micronucleus formation is an important
component of toxicology evaluation of new drug candidates
and other chemicals and materials, such as food dyes and
cosmetics that are intended for human consumption or use, or
which may be indirectly or accidentally consumed or ingested.
Micronuclei formation occurs during cell division of cells
exposed to genotoxic compounds either as a result of DNA
strand breakage (clastogenic compounds) or through
interference with chromosome segregation (aneugenic
compounds) by interference with components of the cell’s
chromosome separation machinery, such as tubulin (Fig 12).
Manual scoring of micronucleus assays is time consuming and
subject to operator variance, bias, and error. Automated
analysis of micronucleus assays allows significantly faster
analysis and consistently objective scoring.
The IN Cell Analyzer Micronuclei Formation Analysis Module
enables fast automated scoring of micronucleus assays. The
software allows the user to set parameters to identify nuclei,
segregate mono-nucleate and bi-nucleate cells (for cytokinesis
block protocols) based on nuclear DNA content and symmetry,
and to define a search area around each nucleus to identify
micronuclei (Fig 13).
The software is compatible with either single-channel imaging
(DNA staining only) or with two-channel imaging (DNA and
cytoplasm staining). Additionally the software provides the
option to use a third imaging channel in combination with
live-cell staining to detect and reject cells with damaged
cytoplasmic membranes from assays where cytotoxicity
is present.
In a typical cytokinetic block assay, exposure of cells to
increasing concentrations of compounds of known
genotoxicity results in an increase in the percentage of binucleate cells with micronuclei (Fig 14A). As cells are exposed
to higher doses of compounds, cell cycle inhibition and
cytotoxicity results in cell arrest prior to mitosis. This prevents
micronuclei formation, with a resulting drop in micronuclei
frequency at higher compound doses (Fig 14B).
Fig 12. Micronucleus formation during cell division.
Fig 13. Identification of micronuclei using the
Micronucleus Formation Analysis Module.
(A) Hoechst stained nuclei (B) Segregation of
bi-nucleate [B] and mono-nucleate cells [M],
(C) Search boundaries used for detection of
micronuclei (D) Micronuclei outlined in white.
Fig 14a. Micronucleus assay dose-response
curves. CHO-K1 cells were exposed to increasing
concentrations of clastogens (Mitomycin C and
Bleomycin) and aneugens (Etoposide and
Diethylstilbestrol) and micronuclei measured by
automated analysis.
Fig 14b. Micronuclei assay proliferation
indices reporting the ratio of bi-nucleate to
mono-nucleate CHO-K1 cells exposed to
increasing concentrations of clastogens and
aneugens. Proliferation index measured by
automated analysis.
Intracellular signaling integrity
In addition to effects on the physical integrity of cells,
candidate drugs may also interfere with essential cell signaling
pathways. To allow evaluation of possible interactions with key
intracellular signaling pathways GE Healthcare has developed
an extensive range of GFP translocation and nitroreductase
(NTR) live-cell reporter gene assays packaged ready to use in
adenoviral vectors. Ad-A-Gene Vectors are validated for
function, provided in a convenient, ready to use format, and
give high-efficiency transduction in both established and
primary cell types (Fig 15).
Used alone or in combination with other cell integrity readouts
in HCA, Ad-A-Gene Vectors provide a powerful toolbox for
detailed investigation of toxic effects of candidate drugs on
cellular integrity (Fig 16).
For further details of Ad-A-Gene Vectors and signaling
pathway coverage, visit www.gehealthcare.com/ad-a-gene.
Fig 15. Cellular transduction with Ad-A-Gene Vectors.
Fig 16. Anisomycin-induced translocation of
GFP-MAPKAP-k2 fusion protein delivered to
HeLa cells with Ad-A-Gene Vector.
IN Cell Investigator Software
The IN Cell Investigator software suite provides a comprehensive
solution to high-content image and data analysis by combining
the latest versions of IN Cell Developer Toolbox and IN Cell Analysis
Modules with Spotfire™ DecisionSite™ visualization software.
Investigator Analysis Modules are a range of preconfigured,
fully validated, and quantitative image analysis routines that
generate statistically relevant data for over 50 applications.
The modules are straightforward to use and deliver the most
relevant measurements for the majority of assays including cell
integrity assays. Simply select the analysis required and start
work. You can either choose specific packages to suit your
unique requirements or combine multiple packages.
Investigator Developer Toolbox is designed for specialized highcontent analysis applications where predeveloped image
analysis is not suitable. The controlled and fully supported
environment helps biologists to build tailored, custom routines
enabling the user to rapidly analyze and interpret results of
complex and unique assays. A selection of advanced
segmentation, preprocessing, and post-processing tools provides
full control over the sequence of steps in analysis routines.
Together these image analysis options provide a wealth of
multiparametric phenotypic data that provide deep insight into
the cellular integrity on many levels.
Spotfire DecisionSite is a powerful data analysis package that
enables rapid interactive visualization, filtering, and sorting of
high-content data. This allows the scientist to explore in-depth
changes to the cellular integrity in response to cellular stimuli
and perturbation.
IN Cell Translator
IN Cell Translator is an optional software tool to convert image
data from other high-content imaging systems to the IN Cell
Analyzer 1000 and 3000 format. This conversion allows the
analysis of images from other platforms with IN Cell Investigator
software. Please contact us for a full list of compatible formats.
Fig 17. Spotfire DecisionSite 3D scatterplot
of data from a siRNA screen. Data for cell
number, nuclear area, and the
nuclear/cytoplasmic distribution ratio of
the G1S Cell Cycle Phase Marker EGFP
fusion protein are shown as SD from
mean for each siRNA knockdown. Data
points are additionally coded for
nuclear/cytoplasmic distribution by color,
and for nuclear area by size.
Products for cellular integrity assays
GE Healthcare has developed a range of assays, reagents, and image analysis software that can be used to assess the
effects of candidate drugs on cellular integrity.
Assays and reagents
Product
Pack size
Code number
5 × 107 ifu
5 × 107 ifu
5 × 107 ifu
5 × 107 ifu
5 × 107 ifu
5 × 107 ifu
5 × 107 ifu
5 × 107 ifu
5 × 107 ifu
GDS20008
GDS20004
GDS20002
GDS20011
GDS20009
GDS20003
GDS40001
GDS40002
GDS40003
Ad-A-Gene Vectors
EGFP-Glucocortocoid receptor
SMAD 9-EGFP
PLC-PH domain-EGFP
2×FYVE domain-EGFP
STAT 3-EGFP
SMAD 2-EGFP
CRE-NTR
NFAT-RE NTR
Ubiquitin C-NTR
This is a selection from a range of over 50 targets. Visit www.gehealthcare.com/ad-a-gene for the complete range of
Ad-A-Gene Vectors.
Cell cycle products
G1S Cell Cycle Phase Marker Assay
G2M Cell Cycle Phase Marker Assay
Cell Proliferation Fluorescence Assay
Screening*
Screening*
500 wells
25-9003-97
25-8010-50
25-9001-89
* Research, technology evaluation, and non-profit assays are also available – please inquire.
CyDye™ labeled second antibodies
Anti-mouse IgG Cy2-Linked (from goat)
Anti-rabbit IgG Cy2-Linked (from goat)
Anti-mouse IgG Cy3-Linked (from goat)
Anti-rabbit IgG Cy3-Linked (from goat)
Anti-mouse IgG Cy5-Linked (from goat)
Anti-rabbit IgG Cy5-Linked (from goat)
1 mg
1 mg
1 mg
1 mg
1 mg
1 mg
PA42002
PA42004
PA43002
PA43004
PA45002
PA45004
Image analysis
Product
Code number
IN Cell Investigator Software, 1 license
IN Cell Investigator Software, 1 additional license
IN Cell Investigator Software, 5 concurrent licenses
IN Cell Translator Software
28-4089-71
28-4089-75
28-4089-72
28-4047-40
Asia Pacific
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Fax: +852 2811 5251
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Tel: 01 6935 6700
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www.gehealthcare.com
GE Healthcare Limited
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UK
imagination at work
General Electric Company reserves the right, subject to any regulatory approval if required, to make changes in
specifications and features shown herein, or discontinue the product described at any time without notice or
obligation. Contact your GE Representative for the most current information. © 2006 General Electric Company
- All rights reserved. Redistribution is a trademark of BioImage A/S; CyDye and Cy are trademarks of GE
Healthcare Companies Limited; Hoechst is a trademark of Hoechst AG. Mitotracker is a trademark of Molecular
Probes. Spotfire and DecisionSite are trademarks of Spotfire Inc. Cyanine dyes are manufactured under license
from Carnegie Mellon University under patent number 5268486 and other patents pending. Use of products
containing GFP is limited in accordance with the terms and conditions of sale. GFP Products are developed and
sold under license from: BioImage A/S under patents US 6 172 188, EP 851874 and EP0986753 and other
pending and foreign patent applications. Invitrogen IP Holdings Inc (formerly Vertex Pharmaceuticals and
Aurora Biosciences Corporation) under US patents 5 625 048, 5 777 079, 5 804 387, 5 968 738, 5 994 077, 6 054
321, 6 066 476, 6 077 707, 6 090 919, 6 124 128, 6 172 188, European patent 1104769 and Japanese patent
JP3283523 and other pending and foreign patent applications. Columbia University under US patent Nos. 5 491
084 and 6 146 826. University of Florida Research Foundation under patents US patents 5,968,750, 5,874,304,
5,795,737, 6,020,192 and other pending and foreign patent applications; and Iowa Research Foundation. Rights
to use this product, as configured, are limited to internal use for screening, development and discovery of
therapeutic products; NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS. No other rights
are conveyed. Ad-A-Gene Vectors are sold under license from: Advec Inc. under patent US 6 140 087, US 6 379
943, US 6 756 226, US 6 855 534, and other pending and foreign patent applications. Transgene S.A is sold
under US 6 136 594 for internal research purposes only and not for any clinical, therapeutic, prophylactic,
diagnostic or production use. The NTR Gene Reporter Assay is the subject of patent WO0186348 and other
pending and foreign patent applications, it is developed and sold under license from Cancer Research
Campaign Technology limited and Proacta Therapeutics limited under patents US5633158, US5780585,
US5977065, AU681337, AU725236 and other pending and foreign patent applications. NTR products are sold for
use with CytoCy5S in in vitro gene reporter assays only. Use in any in vivo application in humans or animals is
strictly prohibited. The G2M Cell Cycle Phase Marker Assay is the subject of patent applications AU 2002326036,
CA 2461133, EP02760417.2, IL 160908, JP 2003-534582, and US 10/491762 in the name of Amersham
Biosciences and Cancer Research Technology. The G1/S Cell Cycle Phase Marker Assay is the subject of patent
applications US 60/590814, US 60/645968 and 60/645915 in the name of Amersham Biosciences and
Vanderbilt University. The IN Cell Analyzer analysis modules are sold under license from Cellomics Inc. under US
patent No 6573039, 5989835, 6671624, 6416959, 6727071, 6716588, 6620591 6759206; Canadian patent No
2328194, 2362117, 2,282,658; Australian patent No 730100 European patent 1155304 and other pending and
foreign patent applications.
28-4087-16 AA