Anti cd11a

Peritoneal B1a cells were obtained through negative magnetic-activated cell separation with a cocktail of antibodies depleting other than B1a cells achieving more than 90% purity in isolation (Miltenyi Biotec B.V., Utrecht, The Netherlands). B1a cells were subsequently counted and plated in IMDM medium (Invitrogen, Eugene, OR, USA) supplemented with 10% fetal calfserum (FCS; Bodinco, Alkmaar, The Netherlands, 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 2 mM L-glutamine (all Gibco Invitrogen, Breda, The Netherlands), and betamercapthoethanol (3.57 x 10 -4 M; Millipore, Amsterdam, the Netherlands). Cells were plated in 96-well plate in density of 1x10 6 /mL in 200L of medium and cultured at 37C and 5% CO2 for 48h in presence of 5g of lipopolysaccharide (LPS, isolated from E. coli strain 055:B5, Sigma, St. Louis, MO, USA; LBP from R&D Systems, Abingdon, UK), isotype control (rat IgG2b, eBioscience, San Diego, CA, USA), anti-CD11b antibody (functional grade, eBioscience, San Diego, CA, USA), or anti-CD11a (functional grade, eBioscience, San Diego, CA, USA) in final concentration 10g/mL. After that supernatant was collected and stored at -80C before measurement of IgM antibody by ELISA. Cells were harvested and processed for analysis by flow cytometry and imaging cytometry.