Edta free trypsin

U27 cells (2 × 10⁵ cells/well/mL) were seeded in 6-well plates, and basic medium containing ISO (20 and 40 μM) was added to each well. Annexin V-FITC/PI Apoptosis Detection Kit (Invitrogen, USA)was used to detect the level of cell apoptosis. After 48 h, the medium was collected, and adherent cells were removed using EDTA-free trypsin (Solarbio, Beijing, China). Samples were centrifuged at 1000× g for 5 min at 4 °C. Next, samples were incubated with Annexin V-FITC solution in the dark for 15 min at 25 °C. Then, propidium iodine (PI) solution was added and incubated for 5 min in the dark. FACSVerse flow cytometer25 (FCM, BD Biosciences, San Jose, CA, USA) was used to analyze the number of apoptotic cells, and FlowJo v10.7 software (Tree Star, Woodburn, OR, USA) was used to determine the apoptotic ratio. Quadrant 1 represents early apoptosis, quadrant 2 represents late apoptotic cells, quadrant 3 represents necrotic cells, and quadrant 4 represents live cells. The apoptosis rate was calculated as the sum of the percentages of cells in quadrant 1 section and quadrant 2 section.

The neuronal PC12 cells in the three groups were washed twice with pre-cooled PBS and processed using the Annexin V-APC/7AAD kit (BioLegend, San Diego, CA, USA) following the manufacturer’s instructions. Briefly, the cells were digested with EDTA-free trypsin (Solarbio, Beijing, China). After incubation with stain and binding buffer at room temperature for 15 min in darkness, the mixed cell suspension was transferred to flow tubes. The apoptosis rate was then determined using a flow cytometer (CytoFlex; Beckman Coulter, Inc., Brea, CA, USA).