Peritoneal lavage was conducted at indicated times after injection of 2 ml of 3% thioglycollate medium (Difco) i.p. Neutrophils were collected by from the lavage at indicated times, washed with RPMI 1640 and macrophages were allowed to adhere to 24-well plates in RPMI 1640 medium for 2 h and non-adherent cells were collected. Forty-eight hours after injections peritoneal cells were collected and were used for downstream applications. Thioglycollate elicited macrophages were cultured at 0.5 × 106 cells per well in RPMI-1640 supplemented with 1% serum in a 24-well plate. Macrophages were co-cultured with neutrophils using a 0.4 μm pore transwell (corning life sciences) with neutrophils cultured in the upper chamber. Cells or supernatant were collected 16–24 h after co-culturing.
Bone marrow from MRP8−Bcl2 mice were collected and crushed and layered over 1077 and 1119 histopaque then centrifuged. Cell layers containing monocytes/macrophages or neutrophils were recovered and washed into PBS then fixed for 20 min with 4% PFA at room temperature. Cells were then blocked for 1 h with PBS containing 1% FCS at room temp followed with staining for calreticulin overnight at 4 °C. Cells were washed three times and AF488 secondary was added and incubated at RT for 1 h then washed three times with wash buffer containing DAPI. Cells were then imaged on the confocal microscope.
Bone marrow from MRP8−Bcl2 mice were collected and crushed and layered over 1077 and 1119 histopaque then centrifuged. Cell layers containing monocytes/macrophages or neutrophils were recovered and washed into PBS then fixed for 20 min with 4% PFA at room temperature. Cells were then blocked for 1 h with PBS containing 1% FCS at room temp followed with staining for calreticulin overnight at 4 °C. Cells were washed three times and AF488 secondary was added and incubated at RT for 1 h then washed three times with wash buffer containing DAPI. Cells were then imaged on the confocal microscope.