Cycle detection kit

Human retinal capillary endothelial cells (HRCECs) were acquired from the Cell Bank of Shanghai Academy of Chinese Sciences (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) was purchased from HyClone (Logan, United States), fetal bovine serum (FBS) was bought from GIBCO (Grand Island, United States). Cell Counting Kit-8 (CCK-8) and PBS were obtained from meilun biotechnology co., Ltd. (Dalian, China). ATP Assay Kit, Mitochondrial membrane potential kit and ROS assay kit were purchased from Beyotime Biotechnology (Shanghai, China). Apoptosis Detection Kit and cycle detection kit were provided by KeyGen Biotechnology co., Ltd. (Nanjing, China). Matrigel was provided by Corning (New York, United States). Primary antibody of p-NF-κB, NF-κB, p-P38, P38, BCL-XL, BCL-2 and GAPDH were bought from Cell Signaling Technology (Danvers, United States).

The sample preparation for flow cytometry was performed as we previously reported [61 (link)]. In brief, cells were seeded in 6-well plates and treated with honokiol at indicated concentration and time. For cell cycle assays, cells were collected and resuspended in 75% ethanol at −20 °C overnight, then incubated with RNaseA and propidium iodide from a cycle detection kit (Keygen Biotech, Nanjing, China). For apoptosis assays, cells were digested by trypsin without EDTA and collected, then stained with annexin V-FITC/PI reagent from an apoptosis detection kit (Vazyme, Nanjing, China). For ROS detection, cells were collected after time gradient drug treatment and incubated with cytosolic reactive oxygen probe DCFH-DA (Beyotime, Shanghai, China) or mitochondrial reactive oxygen probe MitoSOX (Invitrogen, Waltham, MA, USA). The fluorescence signals were measured by a flow cytometer (CyFlow Cube 6, Sysmex, Kobe, Japan).