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).