THP-1 macrophages were left unstimulated or stimulated with control/Rv2615c protein for 16h, 24h and 48h. Annexin V-FITC and PI/ TUNEL assay was performed in Flow Cytometer. 1(a) Graphs were plotted for different time points with a) % of annexinV + annexinV/PI dual positive cells and c) % of TUNEL positive cells on y axis and control/Rv2615c protein on x axis. Statistical analysis was done with Student’s t test and results are Mean ± SEM of three independent experiments where * represents comparison with unstimulated cells and *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. 1(b) Dot plot showing gating strategy for cells stained with AnnexinV-FITC and PI staining which were analysed by Flow Cytometer. Plots were generated as annexinV fluorescence (FL1) on x axis and PI fluorescence (FL3) on y axis. Total 10000 events were acquired for each sample and threshold was set at 80000. Cells in lower right quadrant coloured in green depicts percentage of annexinV single positive (early apoptotic cells); cells in upper right quadrant colours in red depicts percentage of annexinV /PI dual positive (late apoptotic cells); cells in upper left quadrant depicts percentage of PI single positive (necrotic cells). 1d) Dot plot showing gating strategy for percentage of TUNEL positive cells following staining with FITC-dUTP and PI to label the DNA breaks. Plots were generated as FITC-dUTP (FL1) on x axis and PI fluorescence (FL2) on y axis. Total 10000 events were acquired for each sample and threshold was set at 80000. Cells in gate P3 shows the total percentage of TUNEL positive cells or cells with DNA breaks.