4.2.2 (e) Detection of activated initiator Caspase9
Total protein was extracted after 24 hours of protein stimulation by lysing THP-1 macrophages using RIPA lysis buffer and a protease inhibitor cocktail (Santa Cruz Biotechnology Ltd.). Equal concentrations of samples were analysed through SDS-PAGE before being transferred onto nitrocellulose membrane. The total protein content of the entire cell lysate was measured. Primary polyclonal antibody to Caspase9 (PAA627Hu03, Cloud-Clone Corp.), as well as GAPDH (Thermo Scientific) as an internal control, were incubated overnight in membranes after blocking for 1h in 5% skimmed milk dissolved in Tris-buffered saline with Tween 20 (TBST) buffer. Membranes were washed in TBST solution before being incubated for 1 hour with a secondary antibody that has been HRP-conjugated. To create the blot, an ECL Chemiluminescence Kit was employed (Thermo Scientific). Using ImageJ software, Western Blotting pictures were also quantified. Each sample’s developed band area was examined, and graphs were drawn as a ratio to the Unstimulated sample.
4.2.2 (f) Detection of activated Caspases 3 and 7
For detection of activation of Caspase 3 and 7 which is a distinctive feature of early stages of apoptosis, CellEvent Caspase-3/7 Green Flow Cytometry Assay kit (Invitrogen) was used. Following incubation, unstimulated cells and cells stimulated with either Rv2615c or controls- (LPS/Staurosporine) were harvested and 500nM cell event Caspase 3/7 reagent was added to 1ml cell suspension in 1xPBS. Cells were incubated for 30 mins at 37°C. During final 5 mins of staining, 1µM SYTOX dead cell stain was added and samples were analysed in BD accuri C6 flow cytometer.
To confirm the observed apoptosis in response to Rv2615c to be Caspase dependent, Caspase inhibition studies were done. For inhibition study, cells were incubated with 20μM total Caspase inhibitor (Z-VAD-fmk; Promega) for 1 h, followed by treatment with respective test protein/controls and incubated for 24 h. Percentage of Caspase 3 and 7 activation and percentage of AnnexinV and AnnexinV/PI dual positive cell population were analysed using flow cytometry.
4.3. Role of Rv2615c in immune response modulation via expression of TLR, HLA-DR and downstream effector molecules