Anti mouse igg1 antibody

To determine the immune cell subsets in lungs of infected mice, whole lung tissues were removed, followed by isolation of single cell suspensions using the lung dissociation kit (Miltenyi Biotec). Cells were harvested and suspended in FACS buffer (2% FBS in PBS) at a density of 10⁶/ml. The antibodies used in this study are anti-mouse B220 antibody (BD, clone RA3-6B2), anti-mouse IgG1 antibody (BD, clone X56), anti-mouse CD38 antibody (Biolegend, clone 90), anti-mouse CD45 antibody (BD, clone 30-F11), anti-mouse CD49b antibody (Biolegend, clone DX5), anti-mouse CD3e antibody (BD, clone 145-2c11), anti-mouse Siglec-F antibody (BD, clone E50-2440), anti-mouse CD64 antibody (BD, clone X54-5/7.1), anti-mouse CD11b antibody (BD, clone M1/70), anti-mouse CD11c antibody (Biolegend, clone N418), anti-human CD11b (BD, clone ICRF44) and anti-human CD66b (BD, clone G10F5). Cellular fluorescence intensity was analyzed by FACSCanto (BD Biosciences) and FCS Express 3.0 software.

Serum levels of total and HDM-specific IgG1 were determined by ELISA. Briefly, microtiter plates were coated with an anti-mouse IgG1 antibody (0.2 µg/well in PBS, pH 7.4, BD Biosciences) or HDM extract (0.25 µg Der p 1/well in 0.1 M bicarbonate buffer, pH 9.6) and blocked with PBS containing 1% bovine serum albumin (BSA). Serums diluted in PBS containing 1% BSA were then incubated overnight at 4 °C. Next, the plates were incubated with a biotinylated anti-mouse IgG1 antibody (BD Biosciences), an extravidin-horseradish peroxydase (Sigma-Aldrich) and the horseradish peroxydase substrate tetramethylbenzidine (TMB, BD Biosciences), successively. Sample absorbance was measured at 450 nm.