Cell cycle distribution was detected by the classical propidium iodide (PI) staining method and flow cytometric analysis. The cells were seeded in 6-well flat-bottomed plates at 3 × 105 cells per well for 24 h in normal culture medium, and then another 24 h in serum-deprived medium to synchronize cells at G0-phase. After that, the cells were exposed to the selected hydrolysates at 0.05 mg/mL for 48 h. The cells without hydrolysate treatment were served as the control. All cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS) twice, suspended in chilled 70% ethanol at 4 °C overnight, and then re-suspended in the solution containing 50 μg/mL PI, Triton X-100 (0.1%) and RNase A (20 μg/mL) for 30 min at 37 °C in a dark room. For each experiment, 3000 events per sample were recorded by the flow cytometry (FACS Calibur, Becton Dickson, San Jose, CA, USA). Proportion of the cells in G0/G1-, S-, and G2/M-phases were analyzed by the ModFit software (Verity Software House, Topsham, ME, USA).
Two fluorescent dyes, Annexin V-FITC and PI, were used to detect the apoptotic and necrotic cells. Annexin V-FITC identifies early and late apoptotic cells, while late apoptosis and necrotic cells are stained by PI. The protocol used in the present analysis was recommended by the kit manufacturer. Briefly, the experiment was divided into two groups: apoptotic prevention group and apoptotic reversal group. In apoptotic prevention group, the cells were grown to about 80% confluence in the 6-well plates and treated with the hydrolysates for 48 h, followed by 24 h treatment with the proapoptotic agent EP (10 mg/L) and NaF (40 mg/L), respectively. While in apoptotic reversal group, two proapoptotic agents were added before hydrolysates. Those cells without any treatment served as the control group. After treatment, the cells in each group were harvested by trypsinisation, washed twice with PBS and re-suspended in 200 μL binding buffer containing 10 μL Annexin V-FITC and 5 μL PI. The samples were incubated in the dark at room temperature for 15 min, and then analyzed on the flow cytometry. The number of intact cells, early and late apoptotic cells and necrotic cells were discriminated by counting the cells directly.
Two fluorescent dyes, Annexin V-FITC and PI, were used to detect the apoptotic and necrotic cells. Annexin V-FITC identifies early and late apoptotic cells, while late apoptosis and necrotic cells are stained by PI. The protocol used in the present analysis was recommended by the kit manufacturer. Briefly, the experiment was divided into two groups: apoptotic prevention group and apoptotic reversal group. In apoptotic prevention group, the cells were grown to about 80% confluence in the 6-well plates and treated with the hydrolysates for 48 h, followed by 24 h treatment with the proapoptotic agent EP (10 mg/L) and NaF (40 mg/L), respectively. While in apoptotic reversal group, two proapoptotic agents were added before hydrolysates. Those cells without any treatment served as the control group. After treatment, the cells in each group were harvested by trypsinisation, washed twice with PBS and re-suspended in 200 μL binding buffer containing 10 μL Annexin V-FITC and 5 μL PI. The samples were incubated in the dark at room temperature for 15 min, and then analyzed on the flow cytometry. The number of intact cells, early and late apoptotic cells and necrotic cells were discriminated by counting the cells directly.
Free full text: Click here
