Cell Cycle Position Reporting using IN Cell Analyzer Systems
*Kathryn Lamerton, Nick Thomas, Nick Arini, Hayley Tinkler, Paul Michael
GE Healthcare, The Maynard Centre, Whitchurch, Cardiff, UK, CF14 7YT. Tel: +44 (0)2920 526051; Fax: +44 (0)29 2052 6230; E-mail: [email protected]
Introduction
Background
Results
The cell cycle is of key importance to many areas of
The G2M cell line utilises GFP technology to interrogate
Figure 2 depicts an image from the IN Cell Analyzer
drug discovery. On the one hand this fundamental
the position and level of the Cyclin B1-GFP reporter to
1000 showing the GFP G2M cell line with Cy 5 labelled
process provides the opportunity to discover new
determine the cell cycle status in individual cells (Fig 1).
S phase nuclei.
50
% cells
therapeutic targets for anti-cancer agents, and on
the other hand drugs and targets in other therapeutic
G1
areas must be tested for undesirable effects on the
411 cells
40
30
a 20
Mitosis
cell cycle.
10
0
A cell line has been developed that can dynamically
G1
report the cell cycle position of individual cells. The
S
G2
P
M
G2M Cell Cycle Phase Marker cell line stably
expresses a GFP fusion protein which follows the
Fig 3 a. A full-sized image from the IN Cell Analyzer 1000
showing an asynchronous population of 411 GFP G2M cells with
Cy 5 labelled S phase nuclei.
expression and degradation kinetics of Cyclin B1 (1).
This enables non-destructive, dynamic reporting of
the cell cycle status and allows four stages of the
Fig 3 c. A bar graph showing the percentage of cells in each
phase of the cell cycle (plotted using data from Figure 3 b).
References
cell cycle to be identified: G1/S, G2, prophase and
mitosis. However, because cells remain nonfluorescent between mitosis and G2, resolution of
cells into G1 and S phases is not possible. When
multiplexed with a Cell Proliferation Fluorescence Kit
which is used to identify cells in S phase, cells at all
stages of the cell cycle can be identified.
An IN Cell Analyzer 3000 or an IN Cell Analyzer
1000 can be used to monitor the expression levels
and location of the Cyclin B1-GFP fluorescent
reporter and measure the intensity of nuclearTM
associated Cy 5 fluorescence to enable highthroughput cell cycle classification.
Fig 1. Appearance of GFP G2M cells at each phase of the cell
cycle.
The Cyclin B1-GFP reporter 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 nonfluorescent.
0
200
400
600
800
1000
1200
0
1.
Thomas, N and Goodyer, I.D., 2003. Stealth
sensors: real-time monitoring of the cell cycle.
Targets, 2 (1) pp26-33
200
400
The Cell Proliferation Fluorescence Kit 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
Late S,
early G2
600
G2
S phase
800
Conclusions
incorporated into newly synthesized DNA by cells
entering and progressing through the S phase (DNA
synthesis) of the cell cycle. The incorporated BrdU is
detected with a specific anti-BrdU monoclonal antibody,
followed by a Cy 5 labelled antibody to mouse
Fig 2. GFP G2M cells multiplexed with the Cell Proliferation
Fluorescence Kit. Cells at all stages of the cell cycle are
identified. Nuclei are stained with Hoechst™
nuclear dye (blue). BrdU incorporation in S phase cells is shown
in pink (Cy 5). Cells in G1/S, G2, prophase and mitosis are
identified by the position and level of the Cyclin B1-GFP
reporter.
immunoglobulin, giving a fluorescent signal at sites of
BrdU incorporation. This provides an excellent marker
for identifying cells in S phase, and also for determining
the proportion of cells in this phase of the cell cycle.
Figures 3 a, b and c depict image and analysis data
obtained by multiplexing the GFP G2M cell line with the
Cell Proliferation Fluorescence kit to identify the cell
These two assays can be multiplexed to identify cells at
all stages of the cell cycle, providing a high-throughput
method for determining cell cycle position of cell
cycle position of all cells in an asynchronous population,
and also to determine the proportion of cells at each
The GFP G2M Cell Cycle Phase Marker stable
1000
G1
S
G2
P
M
cell line can be multiplexed with the Cell
Fig 3 b. An X,Y plot with each data point representing a cell in
Figure 3 a. Cell cycle classification is denoted by colour. Cells
in G1 (blue), S phase (red), G2 (green), prophase (yellow) and
mitosis (pink) are identified.
Data from the IN Cell Analyzer 1000 was converted using an IN
Cell 123 converter and analysed using IN Cell 3000 Analyzer
software. A 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
reporter. An 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.
phase of the cell cycle.
populations which is currently performed by FACS and
is not amenable to high-throughput.
This technology is described in international patent application number PCT/GB02/04258. The G2M Cell Cycle Phase Marker Assay is the subject of patent GB 2374868 and patent applications PCT/GB01/04363, US09/967301 and
PCT/GB02/04354 in the name of Amersham Biosciences. BioImage A/S. The G2M Cell Cycle Phase Marker Assay is sold under license from BioImage A/S under patents US 6 172 188, EP 851874, US 5 958 713 and EP 0815257, and under
international patent application PCT/EP01/06848 and other pending and foreign patent applications. Invitrogen IP Holdings Inc. This product is sold under license from Invitrogen IP Holdings Inc (formerly Aurora Biosciences Corporation) under
patents US 5 625 048, US 5 777 079, US 5 804 387, US 5 968 738, US 5 994 077, US 6 054 321, US 6 066 476, US 6 077 707, US 6 090 919, US 6 124 128, US 6 319 969, US 6,403,374, EP 0804457, EP 1104769, JP 3283523 and other
pending and foreign patent applications. Columbia University. The G2M Cell Cycle Phase Marker Assay 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. The G2M Cell Cycle Phase Marker
Assay is sold under license from University of Florida under US patents 5 968 750, 5 874 304, 5 795 737, 6 020 192 and other pending and foreign patent applications. The G2M Cell Cycle Phase Marker Assay is the subject of patent application
PCT/ GB02/04258 in the name of Amersham Biosciences. Cancer Research Campaign Technology Limited. The G2M Cell Cycle Phase Marker Assay is sold under license from Cancer Research Campaign Technology Limited under patent
publication number WO 03/031612 and other pending and foreign patent applications. The IN Cell Analyzer 1000 and associated analysis modules are sold under license from Cellomics Inc. US patent Nos 6573039, 5989835, 6671624,
6416959, 6727071, 6716588, 6620591, 6759206; Canadian patent Nos.2328194, 2362117, 2,282,658; Australian patent No.730100; European patent 1155304 and other pending and foreign patent applications
The CyDye in this product is manufactured by Amersham Biosciences under an exclusive license from Carnegie Mellon University under US patent No. 5268486 and other patents pending. Hoechst is a trade mark of Aventis Pharmaceuticals
Inc, NJ, USA.
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All goods and services are sold subject to terms and conditions of sale of the company within the Amersham group which supplies them. A copy of these terms and conditions are available on request General Electric Company reserves the
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information
This poster was presented at the SBS Conference (Society for Biomolecular Screening), September 11–15 2004, Orlando, Florida, USA.
* To whom all correspondence should be addressed.
Proliferation Fluorescence Assay in a fixed cell
assay format to identify the cell cycle position
of all cells in asynchronous populations.
The signal intensity of GFP and Cy5-localised
BrdU is well preserved in a fixed-cell format
and quality data can be obtained from fixed-cell
plates stored at +4oC for up to 1 week (data not
shown).