Allophycocyanin conjugated anti igg1 clone m1 14d12

Resting splenic B cells were collected by negative selection with anti-CD43 and anti-CD11b magnetic beads (Miltenyi Biotec) and cultured in RPMI media (Invitrogen) containing 10% (v/v) fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin-streptomycin (Invitrogen), 2 mM glutamine (Invitrogen), and 50 μM β-mercaptoethanol (Sigma-Aldrich). Cells were plated at 0.5 × 10⁶ cells/ml in 24-well plates and stimulated with 5 μg/ml LPS (E. coli serotype 0111:B4; Sigma-Aldrich) to induce IgG3; LPS plus 5 ng/ml recombinant IL4 (Biolegend) for IgG1; LPS plus 2 ng/ml TGF-β (R&D Systems) for IgG2b; and LPS plus 25 ng/ml IFN-γ (R&D Systems) for IgG2c. Flow cytometry analysis of switched populations was conducted after 4 days using cells stained with FITC- or PerCP-labeled anti-B220 (clone RA3-6B2, eBioscience), and either allophycocyanin-conjugated anti-IgG1 (clone M1-14D12, eBioscience), recombinant PE-conjugated IgG2b, IgG2c, or IgG3 antibodies (Southern Biotech).

B cells were stimulated with LPS as described above and with 0.5 μg/ml anti-mouse CD40 (clone FGK45, Enzo Life Sciences). The following cytokines were then added for specific switching: for IgG1, IL-4 as described above; for IgG2b, 2 ng/ml TGF-β (R&D Systems); for IgG3, no additional cytokines; and for IgA, IL-4, TGF-β, and 1.5 ng/mL mouse IL-5 (R&D Systems). Flow cytometry analysis of switched populations was conducted after 4 days using cells stained with FITC- or PerCP-labeled anti-B220 (clone RA3-6B2, eBioscience), and either allophycocyanin-conjugated anti-IgG1 (clone M1-14D12, eBioscience), recombinant PE-conjugated IgG2b, IgG3, or IgA antibodies (Southern Biotech). For germline transcripts, B cells were stimulated 4 days as described above. mRNA was harvested and converted to cDNA as stated for AID qPCR. PCR was performed as given previously (17 (link), 18 (link)) using primers synthesized by IDT.