Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit

To analyze the cell cycle distribution, MDA-MB-231 (1 × 10⁵) cells were seeded into 6 cm culture dishes. After attaching overnight, cells were treated with the vehicle or ISL at 25 and 50 μM in DMEM/F12 medium containing 10% FBS for 48 h. At the end of incubation, cells were detached by trypsinization. For the cell cycle distribution assay, the cells were fixed in 70% alcohol at −20 °C and stained with a propidium iodide (PI) solution (2 mg of DNAse-free RNAse A and 0.4 mL of 500 μg/mL PI was added to 10 mL of 0.1% Triton X-100 in phosphate buffered saline (PBS)) at room temperature for 30 min. For the apoptosis analysis, a commercial annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was used. The procedure was performed according to the manufacturer’s protocols. The cell cycle distribution and apoptosis were analyzed using a BD FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). A minimum of 10,000 cells per sample were collected and further analyzed using CellQuest software (BD Biosciences).

Purified Colchicine (99.89%), MTT, and DMSO were purchased from Sigma–Aldrich Co. (St. Louis, MO, U.S.A.). Antibodies against Bcl-2, Bax, cleaved-caspase-3, p53, Rb, phospho Rb (pRb), β-actin and horseradish peroxidase (HRP) secondary conjugated antibodies were obtained from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, U.S.A.). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Cell Signaling Technology (Danvers, MA, U.S.A.). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit and propidium iodide (PI) were purchased from BD Bioscience (San Jose, CA, U.S.A.).

For cell apoptosis analysis, GBC-SD and NOZ cells were detected using an Annexin V/fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences), according to the manufacturer's instructions. Human GBC-SD and NOZ cells were harvested following treatment with 25 and 50 µM lenvatinib for 48 h. The cell suspension was transferred to a culture tube and 5 µl propidium iodide (PI) fluorescent dye was added. Cells were then incubated at room temperature for 15 min in the dark, and 400 µl binding buffer was then added. Flow cytometry results were analyzed using a FACS Calibur system (BD Biosciences). For cell cycle arrest analysis, human GBC-SD and NOZ cells were collected following treatment with lenvatinib, washed with cold PBS and fixed in cold 75% ethanol at 4 °C overnight. Subsequently, GBC-SD and NOZ cells were washed with cold PBS, incubated with RNase and stained with PI in the dark for 15 min. The proportion of cells at the G0/G1, S and G2/M phases was determined using the FACS Calibur system, according to the manufacturer's instructions. Data were analyzed by BD FACS Diva 8.0.1 software.