Cell cycle analysis kit

Treated cells were collected and fixated in ice-cold 70% ethanol overnight. Cell Cycle Analysis Kit (Yeasen, Shanghai, China) was used to stain for subsequent flow cytometry. All data were analyzed using Modfit LT 5 (Verity Software).

2 × 10⁵ population of gastric cancer cells were seeded into 12-well plates. After being dealt with the indicated treatment for 24 h, cell suspensions were prepared, and Cell Cycle Analysis Kit (Yeasen) was used to analyze the percentages of cell cycle distribution according to the manufacturer’s instruction.

Apoptosis rates were determined by Annexin V-647 and propidium iodide staining and cell cycle was detected by RNase A and propidium iodide staining according to the instructions of a Cell Cycle Analysis kit (yeasen). The percentage of CD14 and CD11b positive cells was examined by flow cytometry after the treatment with all-trans retinoic acid (ATRA; Sigma) or phorbol 12-myristate 13-acetate (PMA; yeasen). Flow cytometry was performed on a BD cytometer, and the data were analyzed with the FlowJo VX software.

A549 cells at a density of 1 × 10⁵ cells/mL were seeded into wells of the 6-well plates, cultured overnight, and then pretreated with 100 nM KZ-001 for 1 h. The vehicle control group was treated with a 0.5% DMSO culture medium. Then, these pretreated cells were exposed to IR at a 6 Gy dose. After 48 h incubation, the cells were digested and washed with PBS, fixed in 70% ice-cold ethanol at 4 °C overnight, and then processed in compliance with the instructions accompanying the cell cycle analysis kit (Yeasen, 40301). Ultimately, the fluorescence intensity of each sample was measured using flow cytometry (BD Bioscience, FACSCelesta) and analyzed with ModFit LT software.

Flow cytometry-based apoptosis analysis was conducted by Annexin V-FITC cell apoptosis analysis kit (Sungen Biotech, Tianjin, China) and cell cycle analysis was carried out using a cell cycle analysis kit (YEASEN, Shanghai, China). Samples were visualized by FACSCalibur (BD Biosciences, USA) and data were analyzed with FlowJo software (Tree star Inc, CA).

AnnexinV-647 and PI detection kit (Yeasen, Shanghai, China) was used to detect cell death. Cells were collected and washed with PBS and then resuspended in 1× binding buffer. Cells suspension (100 μL) was then transferred to a 5-mL culture tube and stained with 5 μL Annexin V-647 and 10 μL propidium iodide (PI) for 15 min at room temperature in dark. After addition of 200 μL binding buffer into each tube, the apoptotic cells were quantified using Accuri C6 flow cytometer (BD Biosciences, East Rutherford, NJ, USA). Cell Cycle Analysis Kit (Yeasen, China) was used to analyze DNA content and cell cycle profile. The cells were collected and washed with PBS and then placed in 70% ethanol overnight. Cell suspension (100 μL) was then transferred to a 5-mL culture tube and stained with 10 μL PI and 10 μL RNase for 30 min at room temperature in dark. After addition of 200 μL binding buffer into each tube, the apoptotic cells were quantified using Accuri C6 flow cytometer (BD Biosciences, East Rutherford, NJ, USA). Intracellular ROS generation was estimated using DCFH-DA (Yeasen, China). After cells were incubated with 10 μM DCFH-DA for 30 min and washed with PBS, measurements were performed using Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The ROS level is proportional to the mean fluorescence intensity (MFI) of a fluorescent probe DCFH-DA.