Igm clone 2 41

Citrated sheep red blood cells (SRBCs) (Colorado Serum Company) were washed twice with phosphate-buffered saline (PBS) and resuspended in PBS to a final concentration of 10% (vol/vol). Animals were injected intraperitoneally with 0.2 mL of SRBC suspensions. Sera were collected on day 0 and day 7 post-immunization for measuring the levels of SRBC-specific IgM and IgG1 levels using a flow cytometry-based method. Briefly, SRBCs were incubated with varying dilutions of the serum samples, washed and detected with fluorescent conjugated antibodies against either IgM (clone II/41, eBioscience) and IgG1 (cloned A85.1, BD Biosciences). Mean fluorescence intensities (MFI) of the SRBC-bound αIgM and αIgG1 antibodies were plotted against the dilution factors, and the values in the linear range were used for comparing the relative antibody titers.

Bone marrow and spleen single cell suspensions were stained for dead cell exclusion using Live/Dead fixable violet dead cell staining kit (Invitrogen Life Technologies, Grand Island, NY). Surface markers were stained with a mixture of fluorochrome-conjugated antibodies, which included: CD19 (clone 6D5, BioLegend, San Diego, CA), B220 (clone RA3-6B2, eBioscience, San Diego, CA), CD138 (clone 281–2, BD bioscience, San Jose, CA), IgD (clone 11.26c.2a, BioLegend, San Diego, CA), IgM (clone II/41, eBioscience, San Diego, CA), IgG (eBioscience, San Diego, CA), MHC class II (clone 500A2, eBioscience, San Diego, CA), CD80 (clone I6-10A1, BD bioscience, San Jose, CA) CD86 (clone GL-1, BioLegend, San Diego, CA).
Fluorescence minus one (FMO) controls were included in each staining protocol and used to set specific gates (22 (link), 26 , 27 (link)). 6-peak validation beads were used for calibration during the time course analysis. Samples were run on a 12-color LSRII cytometer (BD bioscience, San Jose, CA) and analyzed by Flow Jo software (Tree Star Inc., Ashland, OR).