Vybrant dye cycle violet sytox aadvanced apoptosis kit

Caco-2 cells (2.2 × 10⁵) were seeded onto 21.5 cm² dishes (Nunc International) and then exposed to InsP6 at the concentrations of 1 mM, 2.5 mM and 5 mM for 48 h. Floating and adherent cells were harvested by trypsinization, washed twice with Hank’s Balanced Salt Solution (HBSS, Sigma Aldrich). Pelleted cells were resuspended in 1 mL of HBSS and then stained with the use of Vybrant Dye Cycle Violet/SYTOX AADvanced Apoptosis Kit (Invitrogen, Carlsbad, CA, USA) as described in the manufacturer’s protocol. Viable and dead cells were detected by flow cytometry using BD FACS Aria II detector. The analysis of stained cells allowed to differentiate into four groups, i.e., viable, early apoptotic, late apoptotic and necrotic cells. The apoptotic index, i.e., percentage of early and late apoptotic cells was determined.

CD10^neg/CD10^pos neutrophils (1x10⁶/mL) were cultured for 24 hr in RPMI 1640 medium supplemented with 5% HI-FCS and 1% PenStrep. Apoptosis rate was assessed by flow cytometry using a Vybrant DyeCycle Violet/SYTOX AADvanced Apoptosis Kit (A35135, Invitrogen) and Apotracker Green (Barth et al., 2020 (link)) (427402; Biolegend), according to the manufacturer’s protocols. Stained neutrophils were analyzed using a Gallios cytometer and Gallios software (Beckman Coulter).