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Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000

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.

The Cell Proliferation Fluorescence Kit is a precise, fast, and simple fluorescence assay to quantitate cell proliferation, study the regulation of DNA synthesis, or observe the degree of inhibition of cell progression through S phase. It is based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis in 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 S phase of the cell cycle.

The intensity of nuclear-associated Cy5 fluorescence is measured using an IN Cell Analyzer 1000 and the data analyzed using an Object Intensity Analysis Module.

The use of a Cy5 red-shifted fluor enables this technology to be multiplexed with green fluorescent protein (GFP). This assay provides a high-throughput method for the identification and analysis of cells that have actively synthesized DNA.

Products used

IN Cell Analyzer 1000 25-8010-26

Object Intensity Analysis Module 25-8010-56
for IN Cell Analyzer 1 000

Cell Proliferation Fluorescence Kit 25-9001-89

Other materials required
U2OS (human osteosarcoma) epithelial cells (ATCC, HTB 96) 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)

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 ml
N.B. ensure Triton is properly dissolved before use.

Day 1.

1. Prior to seeding ensure the U2OS cells are sub-confluent.

2. Seed U2OS 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 ste rile 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.

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 ml/well.

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 and 620 nm, emission 535 and 700 nm.

Dichroic D/Cy5. Analyze the images from IN Cell Analyzer 1000 using the Object Intensity Analysis Module to quantitatively measure the nuclear fluorescence intensity per cell.

BrdU incorporation was determined as de scribed in the Method section. Plates were imaged using the IN Cell Analyzer 1000. Figure 1 depicts nuclei of U2OS cells stained with Hoechst nuclear dye (blue). BrdU incorporation is shown in pink (Cy5). Cells in S phase are identified by their pink nuclei.

Figure 2 depicts the effect of Mitomycin C (MMC), a cytostatic drug that inhibits mitosis causing cells to accumulate in G2 phase of the cell cycle. Cells were incubated with increasing concentrations of MMC (as indicated below each set of triplicate images) for 18 h in a 96-well microplate.

Figure 3 is a graphic representation of the fluorescence intensity of each nucleus from the two wells (indicated by arrows) in Figure 2. The Object Intensity Analysis Module was used to derive the nuclear fluorescence intensity (IPos) for each cell (Cell by cell analysis setting). Each data point represents the nuclear fluorescence intensity of one cell. Approximately 250 cells were analyzed per analysis field in each well.

Figure 4 depicts pie charts representing the percentage of cells with a nuclear fluorescence intensity above 300 (these are considered to be in S phase), calculated from data used to plot Figure 3.

Figure 5 shows dose response representing the effect of MMC on the average nuclear fluorescence intensity per well. This was derived using the Object Intensity Analysis Module (Summary setting). Each data point represents the average nuclear fluorescence intensity of three wells. Approximately 250 cells were analyzed per analysis field in each well.

The Cell Proliferation Fluorescence Kit is a sensitive and robust assay that can be used in conjunction with the IN Cell Analyzer 1000 in a fixed-cell assay format to identify cells that are in S phase (Fig 2) and also determine the proportion of cells in S phase (Figs 3 and 4).

The signal intensity of 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).

The assay, in conjunction with automated sub-cellular imaging platforms, is ideally suited for high-throughput primary or secondary screening to uncover novel anticancer drugs and use by toxicologists to establish whether lead compounds adversely effect the rate of cell division.

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