Pi rnase

Cell cycle and apoptosis were analyzed using propidium iodide (1 mg/ml) and ribonuclease-A (10 g/ml) (PI/RNase; BD Biosciences), and Annexin V/PI assay by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) respectively as we previously described (37 (link)).

HT22 cells were plated in 6-well plates at a density of 1.5 × 10⁶ cells per well and treated with drugs for 48 h. Then, cells were trypsinzed and harvested by centrifugation, followed by staining with Annexin V-FITC/PI kit (556547, BD Biosciences, USA); the cells were incubated with Annexin V-FITC and PI at room temperature for 15 min in the dark. The apoptosis rate was examined by flow cytometry (NovoCyte, Agilent, USA) in 30 min. The experiment was repeated three times with four duplicate wells for each group.
Each group of well-grown cells was washed twice with phosphate-buffered saline (PBS), resuspended with pre-chilled in 70% ethanol, fixed at 4° C for 12 h, permeabilized with 0.1% Triton X-100, stained with PI/RNase (550825, BD Biosciences, USA) in PBS, and incubated at 37° C in the dark for 30 min. Subsequently, the cell cycle distribution of each group was detected 3 times by flow cytometry, and the experiment was repeated 3 times.

Cell cycle analysis was performed using flow cytometry (LSRFortessa, BD, USA). The cells were resuspended in 500 µl of PBS (ZLI-9062, Beijing, China) and 3.5 ml of anhydrous ethanol for fixation overnight. Cells (1∗10⁶) were separated by centrifugation at 2,000 rpm for 5 minutes and accumulated and washed twice with cold PBS. Then, 500 µl of PI/RNase (550825, BD, USA) was added to resuspend the cells. After passing through a 200 Mesh Nylon screen, single-cell suspensions were prepared, and the sample was incubated at 4° C in the dark for 30 minutes. Flow cytometry was used to detect red fluorescence at 488 nm and light scattering. DNA content analysis and light scattering analysis were carried out with analysis software. For apoptosis analysis, cells were collected 48 hours after transfection. Cells were double-stained using a FITC Annexin V PE/7AAD Kit (559763, BD, USA). Then, cells were detected by flow cytometry after 30 minutes.

Cell cycle distribution of ACHN and 786-O cells was analyzed through flow cytometry after infection with shRNA lentivirus for 5 days. Then the cells were harvested, washed, and fixed in 75% ice-cold ethanol overnight at −20°C. The cells were then rewashed, stained with PI/RNase (0.5 mL/test, 1 × 106 cells) (BD Pharmingen, United States), and incubated in the dark at room temperature for 15 min before being analyzed by flow cytometry (Guava Technologies; Merck KGaA, Germany). Data were analyzed using ModFit DNA analysis program (Windows version 4.0; Verity Software House).