Pi dye

The BE (2)-M17 human neuroblastoma cell line (NB) and human embryonal kidney cells (HEK293) (5 × 10⁵) were seeded and incubated in the presence of insulin at concentrations of 5, 50, and 100 nM. Forty-eight hours later, the cells were collected and fixed in 4.5 ml cold 70% ethanol. After 2 h, cells were pelleted by centrifugation at 600 × g for 5 min at room temperature (RT). Cell pellets were washed once with PBS and centrifuged at 600 × g for 5 min at RT. Pellets were resuspended in a solution containing 200 μl PI dye (cat No: 75002) (1 mg/ml, biolegend, 421301), 10 mL Triton X-100 (Promega, H5141) and 2 mg RNase (Bioneer, Cat. No. KB-0101). Cells were incubated for 30 min in 37 °C. PI emission was then read at 617 nm wavelength by means of flow cytometry (Partek-Germany).

HaCaT cells were plated at 300,000 cells per well in a 6-well plate for 24 h, and incubated with increasing concentrations of either salpn or DMSO alone for 72 h. Both live and dead cells were collected by centrifugation at 2000 rpm, 4 °C for 5 min. The cell pellets were then resuspended in PBS, washed twice, and resuspended in 100 μl working solution (10 μl of 10 × Binding Buffer, 7 μl AnnexinV-FITC dye, 5 μl PI dye (BioLegend), and 78 μl dH2O). Samples were stained with the Annexin V working solution to distinguish cells that were alive (AnnexinV-; PI-), undergoing early primary apoptosis (AnnexinV+; PI-), and those undergoing late apoptosis/secondary necrosis (AnnexinV+; PI+). The percentage of cells in early or late apoptosis was measured by fluorescence performed on a Cellometer Vision cytofluorometer (Nexcelom). Data shown in each figure represent the mean ± SD of each set of triplicate salpn-treated or untreated cells from a representative experiment.