4.5. Apoptosis and Cell Cycle Analysis Using Flow Cytometry

According to the manufacturer’s instructions, apoptosis was measured using the Annexin V−FITC / PI Apoptosis Detection kit (Elabscience, Houston, TX, USA). BT−20 and MDA−MB−468 cells (1 × 10⁶ cells/well) were treated for 24 and 72 h, respectively, with various concentrations of talazoparib, calcitriol and their combination in a 6−well plate (Based on the IC₅₀ obtained by the RTCA software). The cells were harvested and washed with chilled PBS in a polystyrene round−bottom tube prior to suspension in 100 µL Annexin−binding buffer (ABB). Subsequently, the cells were stained with 2.5 µL of Annexin V and 2.5 µL of propidium iodine (PI) staining solution for 15 min. Staining was performed in the dark at room temperature. A total of 400 µL of ABB was then added to the stained cells prior to analysis with the FACSCanto II flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). For each measurement, at least 10,000 cells were counted.

For cell cycle analysis, BT−20 and MDA−MB−468 with a density of 1 × 10⁶ cells/mL were treated with various concentrations of talazoparib, calcitriol and their combinations for 24 h in a 6−well plate. The treated cells were harvested and washed with chilled PBS, centrifuged (1500 rcf, 7 min), fixed with 70% cold ethanol overnight at 4 °C, and then centrifuged again. Subsequently, cells were washed with PBS to remove excess ethanol and stained with 500 µL of 20 µg/mL PI solution (BD Bioscience) for 30 min. Staining was performed in the dark at room temperature. The cellular DNA contents were identified for detection of the cell cycle distribution using the FACSCanto II flow cytometer (BD Bioscience) installed with ModFit LT (Verity Software House). At least 10,000 events were counted for each sample.