Cxp analysis software

MTP was measured by flow cytometry; 5 μL of 10 μg/mL JC-1 solution was added into 500 μL of cell suspension, mixed, and incubated in dark for 20 min. Stained cells were analyzed by fluorescence activated cell sorting using the CXP analysis software (BD Biosciences, Franklin Lakes, NJ, USA).

Cells were seeded at 350 000 cells per well onto 6-well plates, treated the next day with the compounds as indicated. At each time point, media was then collected from each individual well and cells were trypsinized for 3 min in the tissue culture incubator. Upon centrifugation at 1000 r.p.m. for 5 min, cell pellet was fixed in ice cold 70% ethanol and stored at 4 °C for at least 24 h. On the day of analysis, the fixed cells were treated with RNase A (Invitrogen, Grand Island, NY, USA), stained with Propidium Iodide (PI) (Invitrogen), and analyzed on a Beckman Coulter FC500 Analyzer (Beckman Coulter) using CXP analysis software (BD Biosciences, San Jose, CA, USA). All experiments were performed at least twice independently.

Caki-1 cells exposed to 40 μg/ml EVO for 24, 48, and 72 h were collected at a concentration of 1 × 10⁶ cells/ml. Cells were washed three times using phosphate-buffered saline (PBS). For cell cycle analysis, fixed cells were stained with propidium iodide (PI) solution (20 μg/mL) containing 0.1% Triton X-100 (Sigma–Aldrich, St. Louis, MO, USA) and 100 μg/mL RNase A (Fermentas International Inc., Burlington, ON, Canada). For Annexin V-FITC/PI analysis, cells were stained with FITC-Annexin V Apoptosis Detection Kit I (BD Pharmingen) following the manufacturer’s instructions. A FACScan flow cytometer was used to detect the stained cells (at least 10,000 cells), and CXP analysis software (BD Biosciences, San Jose, CA, USA) was used to analyse the flow cytometry data.