Evaluating Cell Death in Human RPE Cells

TUNEL staining was performed following manufacturer’s instructions (In Situ Cell Death Kit, TMR red; Roche Diagnostics, Indianapolis, IN) as described previously^{32 (link)}. Human RPE was plated on cell culture coverslips (Thermoscientific, Rochester, NY). After treatment, the cells were first fixed in 4% paraformaldehyde for 1 hour at room temperature. After three washes in PBS, cells were incubated with freshly prepared permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 mins on ice. After permeabilization, some cells were incubated with DNase I (3000 U/ml in 50 mM Tris-HCl, pH 7.5, 1 mg/ml BSA) for 10 minutes at 15–25 °C as positive controls. Cells incubated only with Label Solution without Enzyme Solution were used as negative controls. To identify TUNEL+ cells, cells were incubated with TUNEL reaction mixture (Label Solution and Enzyme Solution Mix in 10:1) for 60 mins at 37 °C in a humidified incubator in the dark. After two washes in PBS, cover slips were mounted with DAPI Fluoromount G. Images were taken under confocal microscope with five random images per coverslip. TUNEL+ cells determined by colabeling with DAPI stained nuclei were quantified, and the mean of TUNEL+ cells in the five images from the same coverslip was used for comparison. There were 5-6 coverslips per condition.