Mitochondrial membrane potential kit

Cells were collected for early apoptosis detection based on potential changes in the mitochondrial membrane measured using a mitochondrial membrane potential kit (Cat# C2003S, Beyotime) and fluorescence microscopy.

The mitochondrial membrane potential was assessed using the mitochondrial membrane potential kit from Beyotime Biotechnology (Shanghai, China). Cells were seeded and incubated overnight at 37 °C. Following the instructions provided with the JC-1 reagent kit, JC-1 was diluted 1000-fold to prepare the working solution. Subsequently, 1–2 mL of the working solution was added to fully cover the cells on the culture dish and incubated at 37 °C in the absence of light for 15 min. The cells were then washed three times with PBS buffer. Images were captured using an inverted fluorescence microscope, and changes in the membrane potential of MRC-5 cells were analyzed using ImageJ software.

K562 cells (1 × 10⁶ cells/mL) were inoculated into 24-well plates and maintained for 24 h. Then, cells were treated with AMP-17 (40, 60, 80 μg/mL) and Ara-C (3.5 mg/mL) for 24 h, respectively. After incubation, cells were collected and washed twice with PBS. Each group of cells was added with 0.5 mL of JC-1 staining solution according to the Mitochondrial Membrane Potential Kit (Beyotime, China) protocol and incubated in the dark for 20 min at 37 °C. The samples were measured using a flow cytometry (Beckman, USA).

For cell cycle analysis, cells were harvested, fixed with 75% ethanol for 48 h at 4 °C and stained with propidium iodide (PI; BD Biosciences; Franklin Lakes, NJ, USA) for 15 min. For the detection of apoptosis, cells were rinsed with PBS, resuspended in staining buffer and incubated with Annexin V-FITC and PI (BD Biosciences) for 15 min. The mitochondrial membrane potential was determined with the Mitochondrial Membrane Potential kit (Beyotime). All the cells were assessed on a C6 flow cytometer (BD Biosciences) and the data were analyzed with FlowJo software (V10, BD Biosciences).

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).

Excessive accumulation of ROS is the main cause of oxidative stress, which plays an important role in IDD. So, the intracellular ROS production was assessed using a Reactive Oxygen Species Assay Kit (S0033, Beyotime, Shanghai, China) following the manufacturer’s guidelines. The CEP cells were washed three times with serum-free DMEM/F12 before being treated with TBHP or VO-OHpic. Then DCFH-DA was diluted to 10 uM and added into cells for 30 min in the dark. After washing with serum-free DMEM/F12, the mean fluorescence intensity was calculated using a FACSCalibur flow cytometer, (BD Biosciences, Franklin Lakes, NJ, USA).
MMP decreased in the early stage of apoptosis, the MMP changes were evaluated via mitochondrial membrane potential kit (C2006, Beyotime, Shanghai, China). After washing three times with PBS, the cells were then incubated with the JC-1 staining working solution and equal volume of serum-free medium for 30 min at 37°C in the absence of light. After washing with ice-cold JC-1 washing buffer for 2 times, the cells were analysed using the fluorescence microscope (Axio Observer 3; Carl Zeiss).