Annexin 5 fitc and pi apoptosis detection kit 1

The apoptotic rate of HT22 cells was evaluated by Annexin V-FITC and PI apoptosis detection kit I (BD Pharmingen; BD Biosciences), following the manufacturer's protocol. At 24 h after sevo treatment, HT22 cells were subjected to OGD/R, and then cells were collected, washed with phosphate-buffered saline (PBS) and resuspended in binding buffer, and double-stained with Annexin V-FITC and PI for 10 min at room temperature in the dark. After incubation for 15 min at room temperature in the dark, cell apoptosis was analyzed by FACScan flow cytometer (FCM; Beckman Coulter, Inc.) and CellQuest software version 3.3 (BD Biosciences). The apoptosis rate was the sum of the Q2 and Q3 quadrants of the flow cytometry images.

HSC-T6 cells were treated with OJE (0.1 and 0.5 mg/mL) and silymarin (0.05 mg/mL) for 24 h and harvested for apoptosis assay by using Annexin V-FITC and PI Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Data were analyzed using the BD CellQuest software (BD Biosciences, San Jose, CA, USA), which allowed assessment of specific populations. Individualization by gates was conducted according to size (FSC), granularity (SSC), and fluorescent (FL) parameters. Both early apoptotic (Annexin V⁺ and PI^-) and late apoptotic (Annexin V⁺ and PI⁺) cells were included in cell death determinations.