Annexin 5 propidium iodide double staining

Apoptosis was also evaluated by flow cytometry with Annexin V/propidium iodide (PI) double staining (invitrogen, Eugene, Oregon, USA), according to the manufacturer’s instructions. The analyses were performed by using a guava easyCyte 8HT flow cytometer (Millipore, Billerica, MA).

For cell activity assays, cultured cells were seeded into 96-well plates. After treatment, the cells were incubated with the Cell Counting Kit-8 (CCK8) reagent for 2 h at 37 °C for 5 consecutive days for proliferation analysis or 48 h for IC₅₀ detection. Cell activity was quantified by colorimetric reading in a microplate reader at an absorbance wavelength of 450 nm. For apoptosis analysis, cultured cells were seeded into six-well plates. Cell death was assessed using a Cytotoxicity Apoptosis Detection Kit (Roche, Basel, Switzerland) by flow cytometry with Annexin V/propidium iodide (PI) double staining (Invitrogen). For the colony formation assay, 500 cells were seeded into six-well plates after transfection and cultured in DMEM medium for 10-14 days. The cells were washed with phosphate-buffered saline (PBS) and fixed in methanol for 15 min, followed by staining with 0.1% crystal violet. Cell numbers were counted at the macrographic level. For EdU staining, cultured cells were seeded into 96-well plates after transfection. After 48 h, the cells were fixed in methanol for 15 min, permeated with 0.1% Triton X-100, and blocked with 3% bovine serum albumin before EdU and Hoechst staining. Fluorescence images were captured with a fluorescence microscope (Olympus Corporation, Tokyo, Japan).