2.5. Cell death and cell cycle analysis: Flow cytometry studies

MDA‐MB‐231 and HS‐578T cells (200.000 cells/plate in 60 mm dishes) were treated with olaparib (2.5 and 5 μmol/L, and 25 and 50 μmol/L, respectively), dasatinib (100 and 250 nmol/L, and 50 and 100 nmol/L, respectively) alone or in combination. Final analysis was performed on a FACSCanto™ II flow cytometer (BDBiosciences).

For cell cycle analysis, after 24 hours of treatment, cells were collected, washed with PBS, fixed in 70% ethanol for 30 minutes in ice cold and then, centrifuged at 4000 rpm/5 minutes. Pellets were washed in PBS containing BSA at 2% and incubated with Propidium iodide/RNAse staining solution (Immunostep SL) for 1 hour at 4°C in the dark.

For cell death studies, after 72 hours of treatment, adherent and floating cells were collected, washed with PBS and then, stained with 2.5 μL of Annexin V‐DT‐634 (AV) (Immunostep SL) and 3 μL of Propidium iodide (PI) (10 mg/mL) in 1x Annexin Binding Buffer (10 mmol/L HEPES, pH 7.4, 140 mmol/L NaOH, 2.5 mmol/L CaCl2) for 1 hour at room temperature in dark conditions. Analysis distinguished living cells (AV‐, PI‐) vs apoptotic cells (AV+, PI‐ and AV+, PI+).

For caspase assay, cells were seeded at the conditions described above. MDA‐MB‐231 and HS‐578T cells were treated with olaparib (5 μmol/L and 50 μmol/L, respectively), dasatinib (50 nmol/L and 250 nmol/L, respectively) alone or in combination. Prior to drug exposure, cells were culture in presence or absence of pan‐caspase inhibitor QVD (10 μmol/L). QVD was maintained during all drug treatment.