application note
Cell cycle position reporting
product code:
25-9001-89
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 antiBrdU monoclonal antibody, followed by a Cy™5
an
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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.
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 nuclearassociated Cy5 fluorescence are measured using
an IN Cell Analyzer 1000. The data is converted
using the IN Cell 123 Converter to enable analysis
of the images using IN Cell Analyzer 3000
software. Data is 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
Cell cycle position reporting
Other materials required
McCoys Growth medium:
5A medium modified (Sigma, M-8403) containing:
2% (v/v) Geneticin (Sigma G-7034),
10% (v/v) FBS (Sigma, F-9423),
1% (v/v) L-Glutamine (Invitrogen, 25030-024)
and 1% (v/v) Penicillin/Streptomycin (Invitrogen, 15140-122)
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:
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 µl 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 µl 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 µl/well.
6. Add 200 µl 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 µl/well.
8. Reconstitute Nuclease reagent with 26 ml of sterile water. To
this, add 260 µl of anti-BrdU monoclonal antibody. Add 50 µl
to all wells.
9. Incubate plate for 45 min at 37 °C in 5% CO2.
10. Discard Nuclease reagent and wash the cells three times with
sterile PBS, 200 µl/well.
11. Reconstitute the Cy5 labeled goat anti-mouse reagent with
275 µl of sterile water.
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12. Dilute the reconstituted Cy5 labeled goat anti-mouse reagent
1:100 with sterile PBS. Add 50 µl 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 µl/well.
15. Dilute Hoechst nuclear dye to 2 µM with sterile PBS. Add
100 µl 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 µl/well.
17. Leave the cells in 100 µl 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.
Images were taken using IN Cell Analyzer 1000 using X10 objective
(Fig 2a). Data was converted using IN Cell 123 converter and
analysed 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 Cy 5-localised 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.
Cell cycle position reporting
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.
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.
Fig 2a. An asynchronous population of G2M Cell Cycle Phase Marker stable
cells with Cy5 labeled S phase nuclei.
Fig 2c. Depicts a bar graph showing the percentage of cells in each phase
of the cell cycle (plotted using data from Figure 2b).
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).
The signal intensity of GFP and Cy5-localized BrdU is well preserved
in a fixed-cell format and quality data can be obtained from fixedcell plates stored at +4 °C for up to 1 week (data not shown).
Applications for this high-throughput system include primary or
secondary screening to uncover novel anti-cancer 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.
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Cell cycle position reporting
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shop online www.amershambiosciences.com/incellanalyzer
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 and Amersham Biosciences are trademarks
of Amersham plc. Cy is a trademark of Amersham Biosciences Ltd. 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 815257and 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.
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