4.2.2 (d) DNA Fragmentation analysis by TUNEL Assay
Unstimulated and cells stimulated with Rv2615c or controls- (LPS/Staurosporine) were incubated for different time points. Harvested cells were fixed in 1% paraformaldehyde and incubated for 60 minutes on ice. Fixed cells were washed first with ice-cold PBS followed by 70% ethanol and initially incubated for 30 minutes on ice and later on stored at -20 °C for 18h. Finally, for labelling DNA breaks to detect apoptosis; according to the manufacturer’s instructions, cells were stained with the APO-Direct kit (BD Pharmingen) and analysed using the BD accuri C6 flow cytometer. Briefly, Cells were stained in reaction buffer constituting of Terminal de-oxynucleotidyltransferase (TdT) enzyme and fluoresceinated-dUTP (FITC-dUTP).