Facs420

GBC cells were cultured, and 1 × 10⁶ cells were collected. Annexin V / propidium iodide (PI) staining procedures were performed according to the following reference [34 (link)]. Apoptosis was detected with flow cytometer (FACS420, BD Biosciences, San Jose, CA, USA). Xenografted tumors in BALB/C nude mice were paraffin-embedded, sectioned and subjected to TUNEL assay (Maxim Biotech Inc., Fuzhou, China) to detect cell apoptosis [33 (link)]. The proportions of positive cells in each slice were counted within five high-power fields under microscope.

Flow cytometry was used to determine the apoptosis rate of neural cells. Ischemic brain tissues of eight mice from each group were rapidly stripped on ice. Single-cell suspensions were prepared mechanically, centrifuged at 4°C and 1,200 × g for 10 min, and then the supernatant was discarded. The cells were suspended in 1 ml cold PBS by gentle shaking, centrifuged at 4°C and 1,000 rpm for 10 min, and then the supernatant was discarded. The cells were resuspended in 200 μl binding buffer and 10 μl Annexin V-fluorescein isothiocyanate (FITC) and 5 μl propidium iodide (PI) were then added. The cell suspension was gently mixed and stored in the dark at room temperature for 15 min. Finally, 300 μl binding buffer was added to the suspension, which was analyzed using flow cytometry (FACS 420; BD Biosciences, Franklin Lakes, NJ, USA).

Cells were cultured in 6-well plates at 1×10⁶ cells/well and harvested. The procedures of propidium iodide (PI) staining, Annexin V/PI staining, and the examinations of cell cycle and apoptosis by flow cytometer (FACS420, BD Biosciences, San Jose, CA) were operated as described previously [43 (link)].

For cell cycle analysis, cells (1×10⁶ cells per well) were seeded into 6-well plates, cultured for 48 h, washed twice with cold PBS, stained with propidium iodide (PI) and Annexin V, then examined cell cycle by flow cytometer (FACS420, BD Biosciences, San Jose, CA) within 2 h.

Annexin V/PI staining was performed according to previous procedures [15 ]. Cells from different groups were collected, centrifuged at 1500×g for 5 min, and washed with PBS for three times. 1× Annexin V binding buffer was added to make a final concentration of 2 × 10⁵/mL. Annexin-V and PI (propidium iodide) solution (100 μg, 1 μg/ml) were added for staining at room temperature for 15 min, then flow cytometry was used to evaluate cell apoptosis. The apoptotic cells were detected by flow cytometry (FACS 420, BD Biosciences, USA). Percentage of apoptosis rate (%) = (number of apoptotic cells/number of all cells) × 100%.

The melanoma cell lines M21 and A375 and their transfected or infected derivatives were cultured for 48 h. After the cells were collected, a portion of the cells was fixed with ice-cold 75% ethanol and incubated in a 4°C refrigerator overnight. Cells were washed twice in PBS and then incubated with a propidium iodide (PI) staining mixture containing RNase for 30 min in dark. The cell cycle status was detected by flow cytometry. The remainder of the cells was stained with Annexin V/PI. Apoptosis was detected with a flow cytometer (FACS420, BD Biosciences, San Jose, CA, USA).

Cells were inoculated onto a 6-well plate at 5 × 10⁵ cells/well. After treatment of Tβ4 (Gift by RegeneRx company) for 48 h, the cells were collected and washed twice with pre-cooled PBS. Cells were fixed in pre-cooled 75% ethanol in a 4°C refrigerator overnight, washed with PBS twice, and stained with propidium iodide (PI) containing RNase in the dark for 30 min. Cell cycle phase was detected using a flow cytometer (FACS420, BD Biosciences, San Jose, CA).