Igg2b r12 3

Reagents were purchased from Biolegend, BD, eBioscience, or Invitrogen except for 5D2 and peptide MHC class I-monomers (pMHC class I-monomers, provided by the NIH Tetramer Core Facility, H-2K(b)/SSIEFARL and H-2K(d)/SYIGSINNI). pMHC-monomers and 5D2 were fluorochrome-conjugated using protein labeling kits per the manufacture's instructions (Invitrogen). For pMHC-monomer and intracellular-IgG⁺ staining, cell surface binding of these reagents was blocked with purified IgG1 and Fc-block (included with cell surface staining). Cells were then fixed with BD Fix/Perm solution. Intracellular staining for pMHC-monomers and anti-IgG subclass antibodies (FITC-conjugated IgG 1, A85-1, IgG2a, R19-15, IgG2b R12-3, IgG3 R40-82, all from BD) was performed in 0.25% saponin, 5% rat serum, in PBS at RT for 1 hr. Sample data were recorded on a LSRII (BD). Counts of BM cells were adjusted for total BM population by multiplication by 14.3 (16 (link)). Analyses were performed using FlowJo software (TreeStar). Gating for all flow cytometry plots was for lymphocytes off a forward/side scatter gate and doublet discrimination. Additional gating is indicated at the top of individual plots.