Cd43 magnetic beads

CD43⁻ cells were isolated from spleens after red blood cell lysis using CD43 magnetic beads and LS columns (Miltenyi Biotec). In vitro B-cell stimulations were performed as previously described^{7 (link)} using Rosa^cre/+Dis3^C/+ and Rosa^cre/+Dis3^C/C CD43⁻ splenocytes. Cre-mediated activation was achieved by tamoxifen treatment (4-OHT: (Z)-4-hydroxytamoxifen; Sigma-Aldrich); FBS (Atlanta Biologicals), LPS from Escherichia coli O111:B4 (Sigma) and IL-4 from Peprotech. Cells were routinely analyzed by flow cytometry to monitor GFP expression, cell activation and viability.

For NB cell adoptive transfers, total splenocytes were harvested from 8–12 weeks old B1–8hi donor mice (Cd45.1 or Cd45.1/2), and mature B-cells were isolated using negative selection with CD43 magnetic beads (130-049-801; Miltenyi Biotec; Somerville, MA, USA). The percentage of NP-binding cells in this population was determined by FC (see below), and a number of mature B-cells corresponding to 2–3×10⁵ NP-binding B-cells was injected i.v. into C57Bl/6J recipient mice (Cd45.2). Recipient animals were immunized with an NP-conjugate 16h after cell transfer, and euthanized for analysis at the stated timepoints.
Antigen-specific MB cells for transfer experiments were generated following the protocol above, and splenic NP-binding MB cells were FACS-sorted three months, or more, after immunization of recipient mice with NP-CGG or NP-KLH. Splenic memory T-cells (MT; CD3+CD4+CD44+CD62L−) were FACS-sorted from OT-II mice, 10 days after immunization with 100μg OVA peptide (#A5503, Sigma-Aldrich; San Luis, MO, USA). For re-call experiments, 8×10³ MB and 4×10⁴ MT cells were injected i.v. into μMT recipient mice (Cd45.2). Recipient animals were immunized with NP-OVA 16h after transfer, and euthanized for analysis 3.5 days after immunization.