Annexin 5 fitc pi apoptosis assay kit

A Cell Counting Kit-8 (CCK-8, Becton, Dickinson and Company, USA) was employed to determine the cell viability. The HMC3 cells were seeded into a 96-well plate, and then were divided into four groups: control group, Exos group, NC mimics group and miR-584-5p mimics group. The cells in the Exos group, NC mimics group and miR-584-5p mimics group were transfected with 10 μg plasma-derived exosomes from sevoflurane-induced POCD patients, NC mimics, as well as miR-584-5p mimics, respectively. Additionally, the HMC3 cells in the control group were without any treatment. After cultured for 24 h, 48 h, and 72 h, 10 μL CCK-8 regent was added to each well, and hatched for 2 h. Finally, a microplate reader (Thermo Fisher Scientific) was used to determine the value of OD_{450 nm}.
After that, the cell apoptosis of HMC3 cells were examined using an Annexin V-FITC/PI apoptosis assay kit (Becton, Dickinson and Company). Briefly, the different cells were harvested, and resuspended in 100 μL 1 × binding buffer. Then, 5 μL Annexin V-FITC and 5 μL PI were added to stain the cells. After incubated in the dark for 15 min at room temperature, 400 μL 1× binding buffer was added, and the cell images were acquired by flow cytometer.

The Annexin V-FITC/PI apoptosis assay kit was used to detect apoptosis according to the manufacturer’s procedure (BD Biosciences, USA). Cells were washed in PBS before being treated with Annexin V-FITC and PI at 37°C in the dark for apoptosis detection (BD biosciences, USA). The flow cytometric analysis was performed for detecting cell apoptosis, and the data were analyzed using Flowjo 10 software (Tree Star Software, USA). The percentage of Q2 + Q3 was used to calculate the apoptosis rate.

To examine the morphology of apoptotic and necrotic cells, the cells were seeded in 6-well plates at ~70% confluency and subjected to the indicated treatments. For flow cytometry analyses, cells were treated with various chemotherapy drugs as indicated. Cells were harvested, washed with PBS three times, and stained using Annexin V-FITC/PI Apoptosis Assay Kit (BD Pharmingen, San Jose, CA, USA) and DCFH-DA Reactive Oxygen Species Assay Kit (Meilunbio, China) according to the manufacturer’s instructions. Stained cells were analyzed with a BD FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ), and the data were processed using FlowJo software.

The digestive enzymes were used to prepare AML cell suspension, and then the Annexin V-FITC/PI Apoptosis Assay Kit (BD-556547, USA) was used to determine cell apoptosis. The results were recorded by flow cytometer and analyzed using Flowjo software.

After the cells were transfected and irradiated, apoptosis was detected using the Annexin V-FITC/PI Apoptosis Assay Kit (BD Biosciences, San Jose, CA, USA) according to the standard protocol. The cells were digested with 0.25% trypsin, and the concentration of the cells to be tested was adjusted to about 5×10^5/ml. 1ml cells were taken, centrifuged at 1000rpm at 4°C for 5 min, and supernatant was discarded. Add 1ml of pre-cooled PBS and gently shake the cells to suspend. The Binding Buffer was diluted 1:3 with sterile deionized water (4 ml Binding Buffer +12 ml sterile deionized water). The cells were resuspend with 250 μl Binding Buffer and the concentration was adjusted to 1×106/ml. Take 100 μl cell suspension into 5 mL flow tube, add 5 μl Annexin V/FITC and 10 μl 20 μg/mL PI solution. Mix well and incubate at room temperature in dark for 15 minutes. Add 400 μl PBS to the reaction tube and analyze by flow cytometry. The blank group and treatment group were set up. The blank control group was negative control without dye treatment, and the treatment group was Annexin V-FITC and PI double staining. Annexin V-FITC and PI positive limits were set in the experimental group according to the negative control group.