Annexin 5 fitc apoptosis detection kit plus

Cell death was evaluated by staining with the Annexin V-FITC Apoptosis Detection Kit Plus (BioVision, Milpitas, CA, USA) and the Hoechst 33342 stain (Thermo Fisher Scientific, Waltham, MA, USA). Huh-7 cells were seeded into a 96-well plate at a density of 1 × 10⁴ cells/mL (200 µL/well), followed by incubation for 24 h and treatment with 1% BSA and 800 μM of PA with or without 200 μM of 5-ALA for four hours. Annexin-V with SYTOX green staining was performed per the manufacturer’s protocol. The plates were analyzed with an All-in-One Fluorescence Microscope BZ-X800 (KEYENCE, Osaka, Japan). Dead cells were quantified by counting Annexin-V and SYTOX Green positive nuclei and Hoechst 33342 positive nuclei in ten random microscopic fields (20×) and the ratio was then calculated.

Caspase activity was measured using the Cell Even Caspase-3/7 Detection Reagent (Thermo Fischer Scientific, Waltham, MA, USA). Cell apoptosis was analyzed by staining with the Annexin V-FITC Apoptosis Detection Kit Plus (BioVision, Milpitas, CA, USA) and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [22 (link),46 (link)]. An all-in-one Fluorescence Microscope BZ-X800 (KEYENCE, Osaka, Japan) was used to acquire the fluorescence images. At least four fields at each condition were randomly captured for quantification.