Pe cy7 labeled streptavidin

Zymosan-induced peritonitis was performed essentially as described (Cash et al., 2009 (link)). In brief, female mice (n = 5–10 per group) at 8–12 weeks of age were injected i.p. with zymosan A particles (250 mg/kg, 0.5 ml saline). Peritoneal lavage fluid was harvested with 5 ml PBS containing 1% FCS at 2 or 6 hr after the injection. Cells and residual zymosan particles were removed by centrifugation at 900×g for 4 min. Mice were grouped randomly, and studied without being blinded.
To detect apoptotic cells, peritoneal cells were suspended in PBS containing 2% FCS, incubated on ice with 1 mg/ml of biotinylated anti-Ly6B.2 (AbD Serotec, UK), followed by the incubation with 2 μg/ml PE-Cy7-labeled streptavidin (BD Pharmingen) and 5 μg/ml FITC-labeled anti-Ly6G (1A8; BD Pharmingen). After washing with Annexin V binding buffer, cells were stained with 2000-fold diluted Cy5-labeled AnnexinV (Biovision, Milpitas, CA) and 500 nM Sytox blue (Invitrogen) and analyzed by flow cytometry.

For surface marker staining, cells were stained in PBS supplemented with 1% FBS for 30 min on ice with the following antibodies (PerCP‐conjugated anti‐CD3, BV421‐conjugated anti‐CD4, PE‐conjugated anti‐CD8, BV570‐conjugated anti‐CD45, BV421‐conjugated anti‐F4/80, BV650‐conjugated anti‐CD86, APC‐conjugated anti‐CD206, PE‐Cy7‐conjugated anti‐CD25, FITC‐conjugated anti‐γδ TCR, APC‐Cy7‐conjugated anti‐CD11b, PE‐conjugated anti‐Ly‐6G, FITC‐conjugated anti‐CD49b, PE‐conjugated anti‐CD19, PE‐conjugated anti‐ST2 antibodies, and APC‐conjugated anti‐NKp46: BD Biosciences, San Jose, CA, USA). After fixation and permeabilization, staining with an APC‐conjugated anti‐FOXP3 antibody (eBioscience) was performed according to the manufacturer's instructions. For IL‐33 staining of primary mouse hepatocytes, cells were stained using a biotinylated anti‐IL‐33 monoclonal antibody (Enzo Life Biosciences, Raamsdonksveer, The Netherlands) and PE‐Cy7‐labeled streptavidin (BD Pharmingen, San Diego, CA, USA). All the flow cytometric data were analyzed and plotted using FlowJo software (TreeStar, Ashland, OR, USA).