( 000 PCell Proliferation F...)Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000

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.

Method
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
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