Annexin 5 fitc and propidium iodide

Apoptotic cells were evaluated using an annexin V-FITC and propidium iodide (PI) (ThermoFisher Scientific, Milan, Italy) kit. Chondrocytes were seeded in 12-well plates (8 × 10⁴ cells/well) for 24 h in DMEM with 10% FBS, before the treatment procedure. Then, the cells were washed and harvested using trypsin, collected into cytometry tubes, and centrifuged at 1500 rpm for 5 min. The supernatant was replaced, and the pellet was re-suspended in 100 μL of 1 × annexin-binding buffer, 5 μL of Alexa Fluor 488 annexin-V conjugated to fluorescein (FITC, green fluorescence), and 1 μL of 100 μg/mL PI (red fluorescence) working solution, incubated at room temperature for 15 min in the dark. Afterward, 600 μL of 1 × annexin-binding buffer was added to the tubes before analysis using a flow cytometer.
A total of 10,000 events (1 × 10⁴ cells per assay) were measured by the instrument, and the results were analyzed with Cell Quest software (Version 4.0, Becton Dickinson, San Jose, CA, USA). Apoptosis analysis was carried out considering the simultaneous staining of cells with Alexa Fluor 488 annexin-V and PI. The results were expressed as the percentage of cells positively stained by each dye (total apoptosis) [69 (link)].

Apoptotic cells were measured by using the Annexin V-FITC and propidium iodide (PI) (ThermoFisher Scientific, Milan, Italy) kit. OA chondrocyte were seeded in 12-well plates (8 × 10⁴ cells/well) for 24 h in DMEM with 10% FBS, before replacement with 0.5% FBS used for the treatment. The apoptosis assay was performed as previously described [69 (link)]. A total of 10,000 events (1 × 10⁴ cells per assay) were measured by the instrument. The results were examined with Cell Quest software (Version 4.0, Becton Dickinson, San Jose, CA, USA). Cells simultaneously stained with Alexa Fluor 488 annexin-V and PI were considered for the evaluation of apoptosis (total apoptosis) [22 (link)]. The results were represented as percentage of positive cells to each dye.

HepG2 cells were plated in a 12-well plate at 4 × 10⁵ cells/well for 12 h. After incubation for 24 h with verapamil dissolved in PBS, Vera@pullulan, and Vera@CLCMP patches at the indicated concentrations, the adherent and floating cells were collected, washed with cold PBS, and stained using the Dead Cell Apoptosis kit with annexin V-FITC and propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA). Data was analyzed using a CytoFLEX benchtop flow cytometer (Beckman Coulter, Fullerton, CA, USA).