Annexin 5 fluorescein isothiocyanate fitc propidium iodide kit

Hispidulin was purchased from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) (0.1%). Bax, cleaved-caspase-3, cleaved-caspase-9, LC3B-I, LC3B-II, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DAPI, DMSO, Dulbecco's modified Eagle medium (DMEM), and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit and terminal deoxynucleotidyl transferase-meditated dUTP-fluorescein nick end labeling (TUNEL) in situ cell death detection kit were from Roche Diagnostics (Indianapolis, IN, USA). The bicinchoninic acid (BCA) protein assay kit was from Thermo Fisher Scientific, Waltham, MA, USA.

The apoptotic rates were analyzed using Flow-Count Fluorospheres (Beckman Coulter, Brea, CA, USA) and an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Roche, Penzberg, Germany) in which annexin V binds to the exposed phosphatidylserine on the plasma membrane of apoptotic cells. The cells were stained according to the manufacturer’s instructions. The percentage of early apoptosis was calculated from the proportion of cells that were annexin V–positive and PI-negative (Q4 in Fig. 1a), while the percentage of late apoptosis plus necrosis was calculated from the proportion of cells that were annexin-V and PI double positive (Q2 in Fig. 1a).Fig. 1

Activation of two-pore domain potassium channels (K2P) improved human retinal pigment epithelia (ARPE-19)-19 cell survival. a Representative annexin V/PI staining of apoptosis cells in riluzole and/or t-BHP treat with ARPE-19 cells for 24.0 h. Graphic representation of the survival ratios for ARPE-19. *P < .05 vs. t-BHP group. b Quantitative analysis of the survival ratio of ARPE-19 at 24.0 h post-treatment. c Quantitative analysis of the early apoptosis ratio of ARPE-19 at 24.0 h post-treatment. *P < .05 vs. t-BHP group