2.8. Apoptosis (TUNEL) Assay

To quantify apoptosis in human stem cell-derived neural cultures infected with rabies virus, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed according to manufacturer’s protocol using the in-situ cell death detection kit, TMR red (Sigma-Aldrich, St. Louis, MO, USA, cat#12 156 792 910). As a positive control for DNA fragmentation, fixed and permeabilized neuronal cultures on coverslips were treated with 3 units of DNaseI (Thermofisher Scientific, Waltham, MA, USA) in 50 mM Tris-HCL for 15 min to induce DNA strand breaks. The cells were then stained with pan-axonal neurofilament antibody to identify neurons. Confocal images of neurons stained with TUNEL TMR red, neurofilament and DAPI were taken with 20× objective covering at least 100 neurons per image. The percentage of TUNEL-positive apoptotic bodies in these images (threshold normalized to controls), relative to DAPI stained nuclei were counted using imageJ (NIH, LOCI, University of Wisconsin, Madison, WI, USA) particle analyzer plugin and watershed function, with the following parameters: size (inch²)—0.003-infinity and circularity—0.00–1.00.