Accuri c5 cytometry

The cells were seeded into six-well plates at a density of 2 × 10⁵ cells/well and treated with (−)-agelasidine A (35, 70, and 140 μM) for 24 h. Caspase-3, -8, and -9 activities were measured using the appropriate CaspGLOW fluorescein active caspase staining kits (Biovision, Milpitas, CA, USA) according to the manufacturer’s protocol and measured by flow cytometry on an Accuri C5 cytometry (BD Biosciences, Ann Arbor, MI, USA).

The release of cytochrome c from the mitochondria to the cytosol was detected by FITC–anti-cytochrome c antibody (Clone 6H2.B4, 1:1000; BioLegend, San Diego, CA, USA). Briefly, cells were seeded into six-well plates (2 × 10⁵ cells/well) and treated with the indicated concentrations of (−)-agelasidine A for 24 h. Subsequently, cells were permeabilized with 100 µL of digitonin lysis buffer (50 µg/mL digitonin and 100 mM KCl in 1× PBS) for 5 min on ice and then fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 20 min at RT. Cells were stained with FITC–anti-cytochrome c antibody in 0.5 mL of labeling buffer (2% BSA in 0.05% Triton X-100 PBS) at 4 °C for 35 min, and then detected for cytochrome c-positive cells by Accuri C5 cytometry (BD Biosciences, Ann Arbor, MI, USA).