Part of GE Healthcare
Application Note 11-0011-46 AB
Cell cycle position reporting
Cell cycle position reporting using IN Cell Analyzer 1000
Introduction
The cell cycle is of key importance to many areas of drug
discovery. On the one hand this fundamental process
provides the opportunity to discover new therapeutic
targets for anti-cancer agents, and on the other hand drugs
and targets in other therapeutic areas must be tested for
undesirable effects on the cell cycle.
Our green fluorescent protein (GFP) G2M Cell Cycle Phase
Marker cell line stably expresses a GFP fusion protein that
follows the expression and degradation kinetics of Cyclin
B1. This enables non-destructive, dynamic reporting of the
cell cycle status in living cells and allows four stages of the
cell cycle to be identified: G1/S, G2, prophase, and mitosis.
However, because cells remain non-fluorescent between
mitosis and G2, resolution of cells into G1 and S phases is
not possible. When multiplexed with the Cell Proliferation
Fluorescence Kit, which is used to identify cells in S phase,
cells at all stages of the cell cycle can be identified.
The Cell Proliferation Fluorescence Kit is designed as a
precise, fast, and simple fluorescence assay to quantitate
cell proliferation and is based on the measurement of
5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA
synthesis of proliferating cells.
BrdU (an analog of the DNA precursor thymidine) is incorporated into newly synthesized DNA by cells entering and
progressing through the S (DNA synthesis) phase of the cell
cycle. The incorporated BrdU is detected with a specific
anti-BrdU monoclonal antibody, followed by a Cy™5
labeled antibody to mouse immunoglobulin, giving a
fluorescent signal at sites of BrdU incorporation. This
provides an excellent marker for identifying cells that are in
S phase, and also for determining the proportion of cells in
this phase of the cell cycle.
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These two assays can be multiplexed to identify cells at all
stages of the cell cycle, providing a high-throughput
alternative to FACS for determining cell cycle position of
cell populations.
GFP fluorescence and the intensity of nuclear-associated
Cy5 fluorescence are measured using an IN Cell Analyzer
1000. The data are converted using the IN Cell 123
Converter to enable analysis of the images using IN Cell
Analyzer 3000 software. Data are analyzed using an Object
Intensity Analysis Module to determine cells in S phase, followed by a Cell Cycle Trafficking Analysis Module to distinguish between cells in G1/S, G2, prophase, and mitosis.
Materials
Products used
IN Cell Analyzer 1000
25-8010-26
Object Intensity Analysis Module
for IN Cell Analyzer 3000
63-0048-93
Cell Cycle Trafficking Analysis Module
for IN Cell Analyzer 3000
63-0050-71
IN Cell 123 Converter
Please inquire
G2M Cell Cycle Phase Marker Assay, Screening
25-8010-50
G2M Cell Cycle Phase Marker Assay, Research
25-8010-51
Cell Proliferation Fluorescence Kit
25-9001-89
Other materials required
9. Incubate plate for 45 min at 37 °C in 5% CO2.
McCoys growth medium:
5A medium modified (Sigma, M-8403) containing:
10% (v/v) FBS (Sigma, F-9423),
1% (v/v) L-Glutamine (Invitrogen, 25030-024) and
1% (v/v) Penicillin/Streptomycin (Invitrogen, 15140-122)
10. Discard Nuclease reagent and wash the cells three times
with sterile PBS, 200 ml/well.
Hoechst nuclear dye (Molecular Probes, 33342)
Triton X-100 (Sigma)
4% Formalin solution (Sigma, HT50-1-2)
PBS (Gibco, 14190-094)
96-well assay plate (Greiner, 655090)
PBS tablets (Sigma, P-4417) used to prepare 2× PBS solution
Fixing solution containing:
4% Formalin solution, 5 ml
Sterile water, 5 ml
2× PBS, 10 ml
Triton X-100, 20 µl
N.B. ensure Triton is properly dissolved before use.
Method
Day 1.
1. Prior to seeding the G2M Cell Cycle Phase Marker stable
cell line (U2OS human osteosarcoma cells), ensure that
the cells are sub-confluent.
2. Seed the cells into a 96-well assay plate at 7000 cells/well
(100 ml aliquots) and incubate at 37 °C overnight in 5%
CO2 together with various dilutions of test substance (e.g.
mitogens cytostatic drugs, growth factors etc.).
N.B. In some cases it may be preferable to add the test
substance on Day 2 and incubate for a shorter length
of time.
Day 2.
3. Dilute BrdU labeling reagent 1:250 with Growth medium
and add 100 ml aliquots to appropriate wells to give a
final dilution of 1:500 in the well.
4. Incubate plate for 1 h at 37 °C in 5% CO2.
5. Discard medium and wash the cells twice with sterile
PBS, 200 ml/well.
6. Add 200 ml of fixing solution to all wells, wrap the
plate in aluminum foil and fix the cells overnight at +4 °C.
Day 3.
7. Discard fixing solution and wash the cells three times
with sterile PBS, 200 ml/well.
8. Reconstitute Nuclease reagent with 26 ml of sterile
water. To this, add 260 ml of anti-BrdU monoclonal
antibody. Add 50 ml to all wells.
11. Reconstitute the Cy5 labeled goat anti-mouse reagent
with 275 ml of sterile water.
12. Dilute the reconstituted Cy5 labeled goat anti-mouse
reagent 1:100 with sterile PBS. Add 50 ml to all wells.
13. Incubate plate for 1 h at room temperature in the dark
(or cover the plate in aluminum foil).
14. Discard Cy5 labeled goat anti-mouse reagent and wash
the cells three times with sterile PBS, 200 ml/well.
15. Dilute Hoechst nuclear dye to 2 mM with sterile PBS.
Add 100 ml to all wells and incubate for 30 min at room
temperature, in the dark.
16. Discard Hoechst nuclear dye and wash the cells twice
with-sterile PBS, 200 ml/well.
17. Leave the cells in 100 ml of sterile PBS. Plates can be
stored at +4 °C (in the dark) for up to 1 week.
Image the fixed-cell plate on the IN Cell Analyzer 1000. Select
the following filter sets for image acquisition: excitation 360,
475 and 620 nm, emission 460, 535 , and 700 nm. Dichroic
D/FITC/Cy5.
Convert the data from IN Cell Analyzer 1000 using an
IN Cell 123 Converter to enable analysis of the images using
IN Cell Analyzer 3000 software. Analyze the data using the
Object Intensity Analysis Module to quantitatively measure
the nuclear Cy5 fluorescence intensity per cell, then analyze
using the Cell Cycle Trafficking Analysis Module to determine
the cell cycle position (G1/S, G2, P, or M) for each individual
cell. Combine object data from both analyses to classify
all cells.
Results
BrdU incorporation was determined as described in the
Method section. Plates were imaged using the IN Cell
Analyzer 1000. Figure 1 depicts nuclei of the G2M Cell Cycle
Phase Marker stable cell line stained with Hoechst nuclear
dye (blue). BrdU incorporation in S phase cells is shown in
pink (Cy5). The Cyclin B1-GFP fusion protein is synthesized
during late S and early G2 phases of the cell cycle. At
prophase it translocates from the cytoplasm to the nucleus,
and at metaphase the cells round up and become intensely
fluorescent. When the cell divides from metaphase onwards,
the Cyclin B1-GFP fusion protein is degraded and the two
daughter cells in G1 are non-fluorescent.
Figure 2 depicts image and analysis data obtained from
multiplexing the G2M Cell Cycle Phase Marker stable cell line
with the Cell Proliferation Fluorescence kit to identify the cell
cycle position of all cells in an asynchronous population, and
also determine the proportion of cells at each phase of the
cell cycle.
Fig 2a. An asynchronous population of G2M Cell Cycle Phase Marker stable
cells with Cy5 labeled S phase nuclei.
Fig 1. G2M Cell Cycle Phase Marker stable cell line with Cy5 labeled S
phase nuclei. Cells at all stages of the cell cycle are identified.
Images were taken using IN Cell Analyzer 1000 using 10 ×
objective (Fig 2a). Data were converted using IN Cell 123
Converter and analyzed using IN Cell 3000 Analyzer software
(Fig 2b & 2c).
The Cell Cycle Trafficking Analysis Module was used to
distinguish between cells in G1/S, G2, prophase, and mitosis
based on the location and abundance of Cyclin B1-GFP
fusion protein. The Object Intensity Analysis Module was then
used to derive the nuclear fluorescence intensity (IPos) of
Cy5-localized BrdU for each cell to determine cells in
S phase. The Cell Cycle Trafficking algorithm was modified
so that cell cycle classification data for each cell could be
output as an X, Y plot (Fig 2b). A bar graph, showing the
percentage of cells in each phase of the cell cycle (Fig 2c)
was plotted using data from Figure 2b.
Fig 2b. X,Y plot with each data point representing a cell in Figure 2a. Cell
cycle classification is denoted by color. Cells in G1 (blue), S phase (red),
G2 (green), prophase (yellow), and mitosis (pink) are identified.
Conclusion
The Cell Proliferation Fluorescence Kit is a sensitive and
robust assay that can be multiplexed with the GFP G2M Cell
Cycle Phase Marker stable cell line in a fixed-cell assay
format to identify the cell cycle position of all cells in
asynchronous populations (Fig 2).
Applications for this high-throughput system include
primary or secondary screening to uncover novel anticancer drugs, toxicological studies to establish whether lead
compounds adversely affect the rate of cell division, and
investigation of the effect of cell cycle position on a
disparate process.
The signal intensity of GFP and Cy5-localized BrdU is well
preserved in a fixed-cell format and quality data can be
obtained from fixed-cell plates stored at +4 °C for up to
one week (data not shown).
Fig 2c. Depicts a bar graph showing the percentage of cells in each phase
of the cell cycle (plotted using data from Figure 2b).
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General Electric Company reserves the right 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.
© 2004 General Electric Company - All rights reserved.
Amersham, Amersham Biosciences and Cy are trademarks of
Amersham plc. Cyanine dye: this product or portions thereof is
manufactured under licence from Carnegie Mellon University
under patent number 5268486 and other patents pending. The
G2M Cell Cycle Phase Marker Assay is developed and sold under
license from: BioImage A/S under patents US 6 172 188, US 5 958
713, EP 851874 and EP 815257 and other pending and foreign
patent applications; and Invitrogen IP Holdings, Inc. (formerly
Vertex Pharmaceuticals (San Diego) LLC 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, 6,319,699 European patent 0804457, 1104769 and
Japanese patent JP3283523 and other pending and foreign
patent applications; and Columbia University. This product is sold
under license from Columbia University under US patent Nos. 5
491 084 and 6 146 826. 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; and University of Florida Research Foundation, Inc.
under patents US patents 5,968,750, 5,874,304, 5,795,737,
6,020,192 and other pending and foreign patent applications; and
Cancer Research Campaign Technology under patent application,
PCT/GB02/004258 and other pending and foreign patent
applications. Amersham Biosciences UK Limited under patent
application GB0307684.1. Amersham plc, a General Electric
company, going to market as GE Healthcare.
11-0011-46 AA