Macs anti mouse cd43

B cells from MD4 or MD4/ML5 mice treated with anti-CD79b mAb or control IgG were enriched by depletion of CD43+ cells with magnetic beads (MACS anti-mouse CD43; Miltenyi Biotec). A total of 10⁶ B cells in 200 ml PBS was adoptively transferred by i.v. injection. One hour later the recipient mice were immunized i.p. with HEL-conjugated SRBCs as described previously (29 (link)).

2 hours before adoptive transfer, C57BL/6 recipient mice received 200 rads irradiation. For MD4 transfers, recipients did not receive prior irradiation. B cells from donor mice were isolated via depletion of CD43+ cells with anti-CD43-conjugated magnetic beads (MACS anti-mouse CD43; Miltenyi Biotec). Alternatively, CD4+ T cells were isolated via CD4 positive selection (MACS anti-mouse CD4 (L3T4) Miltenyi Biotec). Resultant populations were >97% pure based on flow cytometric analyses. Donor B cells were labeled with either CellTrace Violet (Molecular Probes) or CFSE (Molecular Probes) at 5μM for 5 minutes at room temperature prior to transfer. Donor CD4 T cells were labeled with CFSE (Molecular Probes) at 5μM for 5 minutes at room temperature prior to transfer. 1–2×10⁶ donor cells in 200μl PBS were adoptively transferred via IV injection. 24 hours post transfer, Tamoxifen was administered to activate Cre. Tamoxifen (T-5648; Sigma-Aldrich) was dissolved in 100% corn oil (Sigma-Aldrich) at 20mg/ml. Recipient mice were injected IP with 100μl (2mg) on two consecutive days.

Splenocytes were isolated by forcing spleens through 70 micron screens, red blood cells were lysed in a hypotonic solution of ammonium potassium chloride. Cells were washed in IMDM with 2% FCS. In adoptive transfer experiments, “Untouched” B cells were isolated from splenocytes by depletion of CD43⁺ cells with anti-CD43-conjugated magnetic beads as per manufacturers protocol. (MACS anti-mouse CD43; Miltenyi Biotec). B cell purity was routinely >97% and fewer than 1% of these cells were plasmablasts based on B220^low and CD138⁺ phenotype.