Mouse il 6 and tnf α

ELISA kits for mouse IgA and IgM (eBioscience, Frankfurt am Main, Germany), mouse IL-6 and TNF-α (BioLegend, London, United Kingdom), mouse C5a (R&D Systems, Minneapolis, MN) and mouse albumin (Bethyl Laboratories Inc., Montogomery, TX) were used according to manufacturer’s instructions. For SIgA-detection Nunc MaxiSorp plates were coated overnight with monoclonal rat anti-mouse IgA (SouthernBiotech, Birmingham, 2 µg/mL). Plates were washed, blocked and incubated with serial dilutions of BALF samples and incubated with secondary antibody (polyclonal goat anti-mouse pIgR; R&D Systems, 1 µg/mL) followed by polyclonal donkey anti-goat-HRP (Jackson ImmunoResearch, West Grove, PA, 0.16 µg/mL) and TMB substrate. Plates were read at 450 and 570 nm. Due to the lack of a SIgA protein standard, relative quantification was performed. Results are expressed as the OD at 450 nm (wavelength- and blank-corrected) at indicated dilutions.

Cytokine production was quantified using a customized multiplex (Milliplex-MAP, Merck Millipore, France) assay kit and Luminex 100 IS instrument (Luminex, USA) available at the phenotyping service platform Anexplo (CHU Rangeuil, Toulouse, France). Ten cytokines were systematically assayed in peritoneal exudates: IL-1α, IL-1β, IL-6, CXCL1, CXCL2, CXCL5, CCL2, CCL5, BAFF, and TNF-α. For BMDM culture supernatants, individual cytokine-detection kits were used to quantify mouse IL-6, and TNF-α (Biolegend, Ozyme-France).