Isolation and Characterization of Peritoneal B Cell Subsets

Flow cytometric analysis and cell sorting were performed as previously described (36 (link)). Briefly, peritoneal washout cells were obtained from five different mice of each D_H-altered strain. The following mAbs were used to isolate PerC B cells into the B-1a, B-1b, and B-2 subpopulations: anti-B220 (RA 3.6B2) (BD Pharmingen) (Southern Biotechnology, Birmingham, AL, USA), anti-Mac-1 (BD Pharmingen, San Diego, CA, USA) and anti-CD5 (BD Pharmingen, San Diego, CA, USA). PerC B-1a cells were sorted as B220^loCD5⁺, B-1b as B220^loCD5⁻Mac-1^lo/+, and PerC B-2 cells were sorted as B220^hiCD5⁻Mac-1⁻. A MoFlo instrument (Dako) was used for cell sorting.