Ly 6g clone rb6 8c5

Mice untreated or treated with intraperitoneal injection of 1 mL 4% thioglycolate (Sigma-Aldrich) for 4, 24, or 48 h were killed using CO 2 , and peritoneal leukocytes were collected by lavage of the cavity with PBS containing 5 mM EDTA. After centrifugation, cells were incubated 2 min in 1 mL of RBC buffer (170 mM NH4Cl, 12 mM KHCO3, 0.9 mM EDTA, pH 7.6) to lyse residual red cells and, then, centrifugued and resuspended in DMEM and counted using a hemocytometer. For flow cytometry analysis, PerC cells were incubated for 30 min at 4ºC with labeled antibodies against CD19 (clone 1D3, eBioscience), F4/80 (clone Cl:A3-1, AbD Serotec), Ly6-G (clone RB6-8C5, eBioscience), CD5 (clone 53-7.3, Biolegend), CD11b (clone M1/70, BD Pharmingen), and CD3ε (clon 145-2C11, BD Pharmingen), using isotype-matched antibodies as negative control and color compensation as appropriate for dual labeling. Samples were analyzed in a Coulter flow cytometer model FC500. A more detailed analysis of B-cell population was performed by simultaneous labeling with anti-CD19, anti-CD5 and anti-CD11b antibodies. According to the expression of these markers, B cells were considered as belonging to B1a (CD19+/CD5+/CD11b+), B1b (CD19+/CD5-/CD11b+), B1c (CD19+/CD5+/CD11b-) or B2 (CD19+/CD5-/CD11b-) subsets (Tung and Herzenberg, 2007; Rosado et al., 2014; Hastings et al., 2006) .