Fig 1: MIP supernatant induces apoptosis in mouse peritoneal macrophages.(a) MIP supernatant induced cell death of macrophages was not due to LPS contamination. Macrophage monolayers were treated with MIP supernatant (1 µg/ml) for 8 hr and % cytotoxicity was determined by MTT assay. Before incubation of macrophages with MIP supernatant, the supernatant was also treated either with Polymyxin B (PB). Untreated cells were taken as control with 0% cytotoxicity. (b) Murine peritoneal macrophages after treatment with MIP supernatant for 4 hr were dual stained with Sigma APOAC kit. Annexin V stained early apoptotic cells (annexin V positive, 6-CFDA positive) show red fluorescence on cell surface. Untreated viable cells (annexin V negative, 6-CFDA positive) fluoresce green with no signal for Annexin V. (c) Macrophage monolayers were treated with MIP supernatant for 6 hr, fixed & stained for DAPI. Arrows show nuclear condensation & fragmentation. Scale bar: 10 µm. (d) Macrophage monolayers were treated with MIP supernatant for 6 hr, lysed and the DNA fragmentation was detected by quantitative Nucleosome ELISA with anti-histone antibody. Untreated cells were taken as control. (e) MIP cell-free supernatant was lyophilized and re-suspended in sterile PBS & run on SDS-PAGE. The gel was stained with AgNo3 and observed. Lane1: Low molecular weight SDS marker, lane2: MIP supernatant. The indicated molecular weights are in kDa.