Rat anti mouse igm

Immulon 2 plates (ThermoLabsystems, Pittsburgh, PA) were coated overnight at 4 °C with goat anti-mouse human adsorbed unlabeled IgM (Southern Biotech; 100 μl/well). Plates were washed, blocked with 3% BSA in PBS at 37 °C overnight, washed, and 100 μl/well of four two-fold dilutions of sera in PBS + 1% BSA were added starting at 1:2. Sample containing plates were incubated for 1 h at 37 °C and washed. Goat anti-mouse AP conjugated antibodies (Southern Biotech; 100 μl/well) were added for 1 h at 37 °C. Plates were washed and 100 µl of phosphatase substrate tablets (Sigma-Aldrich) dissolved in p-Nitrophenyl Phosphate, Disodium Salt (PNPP) buffer was added to wells. Absorbance was read at 405 nm on an xMark™ Microplate Spectrophotometer with the Microplate Manager™ Software (Bio-Rad, Hercules, CA). Standard curves were generated using serial dilutions of purified rat anti-mouse IgM (Clone II/41, BD), and the 4-parameter fit equation used to calculate sample concentrations.

Antibodies for immunofluorescence studies were as follows: rat anti-mouse IgM (553437, Pharmingen), MOMA-1 (T2011, BMA Biomedicals), MARCO (MCA1849, Serotec), and rat anti-mouse MRP14 (565833, BD Biosciences). Secondary antibodies included goat anti-rabbit FITC-conjugated IgG, rhodamine-conjugated IgG (H + L) (111-225-045, 111-165-144, Dianova), Cy5-conjugated IgG (111-605-144, Dianova), goat anti-rat Alexa Fluor 568 IgG (A11077, Invitrogen), and donkey anti-rat Alexa Fluor 488 IgG, (A21208, Invitrogen).
Antibodies to the following were used in flow cytometry analyses to identify cellular compartments: PE- or APC-labeled anti-mouse Ly6G (127607, 127614, BioLegend), APC-labeled anti-mouse CD11b (17-0112-83, eBioscience), biotin- or PE-labeled anti-mouse CD23 (553139, Pharmingen), FITC- or PE-labeled anti-mouse CD21 (552957, 553818, Pharmingen), FITC-labeled anti-mouse BrdU (559619, BD Biosciences), PerCP-labeled anti-mouse CD19 (552854, Pharmingen), rat anti-mouse MRP14 (565833, BD Biosciences), FITC-labeled anti-mouse BAFF receptor (11-5943, eBioscience), and PE-labeled anti-human BAFF receptor (316906, BioLegend). Human neutrophils and plasma cells were detected using FITC-labeled anti-human CD66b (305103, BioLegend) and APC-labeled anti-human CD138 (352308, BioLegend).