Apoptosis assay and superoxide production in RAW 264.7 cells. (A) Apoptosis assay using annexin-V FITC/PI dual staining method. RAW264.7 were treated with M. koenigii and A. nilagirica extacts for 24 h. The percentage of apoptosis was determined by analyzing 10,000 gated cells using flow cytometry. (B) Intracellular survival of M. smegmatis in A. nilagirica extract treated macrophages. RAW264.7 cells were treated with different concentrations of A. nilagirica extacts 2 h before (Pretreatment) and after (post-treatment) M. smegmatis infection. The cells were lysed and intracellular survival was determined 6 h post infection by CFU assay. Data showed decrease in intracellular bacterial survival in pretreated macrophages. (C) NO production in RAW264.7 treated with different concentrarions of A. nilagirica leaf extract for 24 h (D) ROS production was determined by DCFH-DA staining using flow cytometry indicating increased ROS production. Experiments were performed in triplicates.