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GE Healthcare
Application Note 28-4070-46 AA
IN Cell Analyzer
Micronucleus formation analysis on the IN Cell
Analyzer 1000
Key words: micronuclei genotoxic image
analysis IN Cell Analyzer
IN Cell Analyzer 3000
25-8010-11
IN Cell Investigator Software, 1 license*
28-4089-71
Micronucleus induction is a key characteristic of genotoxic
compounds and analysis of micronuclei formation resulting
from DNA strand breakage (clastogens) or interference with
chromosome segregation (aneugens) is an important
component of toxicology screening of new drug
candidates. Manual scoring of micronucleus assays is time
consuming and subject to operator variance, bias, and
error while automated analysis of micronucleus assays
allows significantly faster analysis and consistently
objective scoring.
IN Cell Investigator Software,
1 additional license
28-4089-75
IN Cell Investigator Software,
5 concurrent licenses
28-4089-72
IN Cell Translator Software
28-4047-40
This application note describes the use of IN Cell Analyzer
1000 Micronuclei Formation Analysis Module for automated
scoring of micronucleus assays. The software is compatible
with standard assay protocols (1,2) and may be used to
analyze images acquired using IN Cell Analyzer 3000 via
image conversion to IN Cell Analyzer 1000 format.
Tissue culture reagents
Other materials required
CHO-KI cells
Imaging grade 96-well plates
(Invitrogen)
(Greiner Bio-One
or Perkin Elmer)
Mitomycin C†
(Sigma, M4287)
Cytochalasin B‡
(Sigma, C6762)
Hoechst 33342
(Molecular Probes)
FITC
Fig 1. IN Cell Analyzer 1000 micronucleus assay. Micronuclei (arrowed) in
CHO-K1 cells exposed to 50-ng/ml mitomycin C for 48 h. (A) DNA (Hoechst)
and cytoplasmic (FITC) staining. (B) DNA staining (Hoechst).
(ATCC or ECACC)
(Molecular Probes, F1906)
*
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.
†
Mitomycin C is classified as toxic; handle in accordance with product
safety data sheets and local laboratory safety guidelines.
‡
Cytochalasin B is classified as very toxic; handle in accordance with
product safety data sheets and local laboratory safety guidelines.
Assay protocol
Materials
Products used
IN Cell Analyzer 1000 end-point instrument
28-4051-28
IN Cell Analyzer 1000 kinetic instrument
28-4051-29
The assay protocol employs a cytokinesis block to ensure
that candidate micronuclei have originated from actively
dividing cells. Cell division is an absolute requirement for
micronucleus formation. Cytochalasin B arrests cell division
immediately following mitosis but prior to separation into
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two distinct daughter cells, at this point, micronuclei will
have formed but cytokinesis (separation of the cell
cytoplasm) is prevented. Consequently, following treatment
with cytochalasin B, cells that have undergone one round
of division are bi-nucleate, and a micronucleus associated
with a bi-nucleate cell can be assumed to have originated
from a single division cycle.
Analysis protocol
1. Seed CHO-K1 cells in 96-well plates at 5000 cells per
100-µl/well and incubate under standard tissue culture
conditions for 24 h.
1. Open run file in the IN Cell Analyzer 1000 analysis
software.
2. Prepare dilutions of test compounds in solvent. Prepare
a 1-mg/ml stock solution of mitomycin C in PBS and
sterile filter for use as a positive control. To each
sample well, add 100 µl of test compounds, solvent,
and mitomycin C (use mitomycin C at 100-ng/ml final
concentration). Incubate cells for 24 h.
3. Select appropriate segmentation settings to identify
nuclei in the blue (Hoechst) channel. Take care to
exclude micronuclei from identified objects.
3. Prepare a 3-mg/ml stock solution of cytochalasin B in
DMSO. Dilute 1:100 in complete tissue culture medium
and add 10-µl/well. Incubate cells for 24 h.
6. Choose segmentation settings to identify micronuclei.
Adjust size and sensitivity parameters if required.
4. Remove media, wash cells once with PBS and fix in
ethanol for 30 min at room temperature.
5. Make 10-mg/ml stock solution of FITC in DMSO. Dilute
10 µl in 100-ml PBS and add 100-µl/well. Incubate for
30 min at room temperature.
The IN Cell Analyzer 1000 Micronuclei Analysis Module uses
a series of operations (Fig 2) based on user settings to
define nuclei, segregate mono-nucleate and bi-nucleate
cells, and define micronuclei (Fig 3). For full guidance on
using the analysis module see the product manual.
2. Select a mitomycin C positive control well.
4. Choose segmentation method and select appropriate
settings to segment cells.
5. Select search region to be analyzed for micronuclei.
7. Generate a sample of cell population and set up
thresholds in the nuclei shape classifier. Adjust
thresholds to segregate mono-nucleate and bi-nucleate
cells. Separate thresholds if required to assign
ambiguous cells as unclassified.
8. Select filters if required.
6. Wash wells three times with PBS.
9. Make choice of measures.
7. Stain cells with 5-µM Hoechst 33342 in PBS for 15 min
at room temperature.
10. Check analysis on second positive control well and
solvent negative control wells. Adjust analysis
parameters as necessary.
8. Image wells using excitation and emission settings for
fluorescein and Hoechst 33342.
Fig 2. IN Cell Analyzer 1000 micronucleus analysis process.
2
Application Note 28-4070-46 AA 2006-04
11. Run analysis.
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A
C
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B
D
Fig 3. Identification of micronuclei using IN Cell Analyzer 1000
Micronuclei Formation Analysis Module. (A) Hoechst 33342 stained
nuclei, (B) Segmented nuclei and cells, (C) Classified bi-nucleate [Bi] and
mono-nucleate cells [Mono], (D) Micronuclei located in cytoplasm are
identified (yellow outline).
Fig 4. Effect of genotoxic compounds on cell proliferation index. CHO-K1
cells were exposed to increasing concentrations of test compound and the
proliferation index was measured by automated analysis.
Fig 5. Micronucleei formation assay dose-response. CHO-K1 cells were
exposed to increasing concentrations of clastogen (mitomycin C ,
bleomycin) or aneugen (etoposide) and micronuclei quantitated by
automated analysis. The percentage of bi-nucleate cells having
micronuclei is plotted as a function of compound concentration.
Data analysis
1. Open data output file and analyze proliferation index
(Fig 4) and micronuclei frequency (Fig 5) data.
2. Discard data from wells where the cell number or the
proliferation index indicate cytotoxic or cytostatic
activity. Compounds showing potential false negative
criteria should be re-tested at a lower concentration.
In assays employing a cytokinesis block, the ratio of binucleate to mono-nucleate cells (proliferation index)
within a given cell population provides an indication of
cell proliferation. The results in Figure 4 demonstrate
dose-dependent decreases in the cell proliferation index
following treatment with known genotoxic agents.
Plotting the percentage of bi-nucleate cells having
micronuclei (Fig 5) reveals that micronuclei formation is
also dose-dependent. The frequency of micronuclei
formation increases with increasing dose until the
concentration of test compound becomes frankly toxic,
at which point the percentage of bi-nucleate cells with
micronuclei begins to decline. When dose-points
corresponding to a low proliferation index (< 50%) are
excluded from the data set, non-linear regression
analysis of the normalized results (Fig 6) enables EC50
evaluation and rank order analysis.
Fig 6. Micronuclei assay dose-response. Data from micronucleus
formation assays (Fig 5) were analyzed by non-linear regression analysis
to determine EC50 values for micronuclei induction. Toxic doses determined
from proliferation index data (Fig 4) were excluded from analysis before
curve-fitting.
Comparison of micronucleus formation
analysis using IN Cell Analyzer 1000 and
by manual scoring
Until recently, micronuclei formation assays were largely
quantitated by manual scoring. Figure 7 compares results
obtained by manual scoring with those obtained using the
automated IN Cell Analyzer 1000 Micronuclei Formation
Analysis Module. Results for scoring micronucleus
formation in bi-nucleate cells using IN Cell Analyzer 1000
Application Note 28-4070-46-00 AA 2006-04
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correlate well with those obtained using manual scoring. In
addition, good correlation between results obtained using
the IN Cell Analyzer 1000 and 3000 Micronuclei Analysis
Modules has been demonstrated (data not shown).
Fig 7. Micronuclei assay mitomycin C dose-response. The number of
bi-nucleate cells with micronuclei was quantitated using the IN Cell
Analyzer 1000 Micronuclei Formation Analysis Module (red line) and by
manual scoring (blue line). Application of the Pearson’s correlation test
demonstrates significant correlation (P = 0.01, r = 0.91, r2 = 0.82)
between the results.
Conclusions
This application note demonstrates use of the IN Cell
Analyzer 1000 Micronuclei Formation Analysis Module to
monitor micronuclei formation in cells assayed using a
cytokinesis block protocol. The Micronuclei Analysis Module
successfully detected and quantitated micronuclei
formation in response to three genotoxic compounds.
Correlation analysis demonstrates that results obtained
using automated analysis agree well with those obtained
by manual scoring. The analysis module can also be used
to analyze micronuclei formation in assays that do not
employ a cytokinesis block. Automated analysis provides
an objective and rapid method for processing large
numbers of cells, improving statistical reliability of the
results and increasing throughput of the assay.
References
1. Kirsch-Volders M. et al. Report from the in vitro micronucleus assay working
group. Mutat. Res. 540 (2), 153–63 (2003).
2. Kirsch-Volders M. et al. Report from the in vitro micronucleus assay working
group. Environ. Mol. Mutagen. 35 (3), 167–72 (2000).
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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. GE and GE Monogram are
trademarks of General Electric Company. Amersham is a
trademark of GE Healthcare Companies. The IN Cell Analyzer
3000 is the subject of US patents 6,400,487 and 6,388,788 and US
patent application number 10/227552, together with other
granted and pending family members, in the name of Amersham
Biosciences Corporation. The IN Cell Analyzer 3000 and
associated analysis modules are sold under license from
Cellomics Inc. under US patent Nos 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. Hoechst is a trademark of Hoechst AG.
Spotfire and DecisionSite are trademarks of Spotfire, Inc.
imagination at work
Application Note 28-4070-46 AA 2006-04