Primary B cells were isolated from spleens of IgHB1-8/B1-8 Igκ −/− transgenic mice (provided by M. Shlomchik, Yale University, New Haven, CT) by negative selection using MACS sorting as described previously (18 (link)). Isolated B cells were cultured overnight with CpG and LPS (Calbiochem, San Diego, CA) and experimented next day. The J558L B cells stably expressing B1-8-γ-cyan fluorescent protein (CFP) and Igα-yellow fluorescent protein (YFP) (γCαY) were established and characterized as previously described (21 (link)). Daudi B cell line stably expressing Lyn16-CFP-YFP fusion protein and CH27 B cell line stably expressing the lipid raft probe Lyn16-CFP, the nonlipid raft probe CFP-Ger, or the full length LynFL-CFP were established and characterized as previously reported (19 (link)). ST486, a human B cell line and A20II1.6, a mouse B cell line, both negative for endogenous FcγRIIB expression, were purchased from the American Type Culture Collection (Manassas, VA). PT67 cell line, a virus packaging cell line, was purchased from BD Clontech (Palo Alto, CA).
Cy3- and Cy5-conjugated Fab goat Abs specific for mouse IgM and IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Cy3-conjugated Fab rat mAb specific for mouse IgM (clone no. II/41) was purchased from Rockland (Gilbertsville, PA). Rabbit IgG Abs specific for BSA (rabbit anti-BSA) were purchased from Bethyl Laboratories (Montgomery, TX) and F(ab′)2 rabbit anti-BSA were prepared as described (20 (link)). PE-conjugated and biotin-conjugated rat mAb specific for mouse FcγRIIB (clone no. 2.4G2) and APC-conjugated mouse mAb specific for human FcγRIIB (clone no. FLI8.26) were purchased from BD Pharmingen (San Diego, CA). Biotin-conjugated mouse mAb specific for human FcγRIIB (clone no. AT10) was purchased from AbD Serotec (Raleigh, NC). Biotin-conjugated F(ab′)2 goat Abs specific for mouse IgG and IgM, biotin-conjugated F(ab′)2 rabbit Abs specific for human IgM, and streptavidin were purchased from Jackson ImmunoResearch Laboratories. Rat mAb specific for mouse FcγRIIB (clone no. 190907) was purchased from R&D Systems (Minneapolis, MN) and Fabs mAb 190907 were prepared using an immobilized papain kit (Pierce, Rockford, IL) following the manufacturer’s protocol. Conjugations of Abs with Alexa514, 568, or 647 were performed using Alexa Fluor mAb labeling kits (Molecular Probes, Eugene, OR) following manufacturer’s protocols. BSA conjugated 1:14 with 4-hydroxy-5-iodo-3-nitrophenyl acetyl (NIP) (NIP14-BSA) and BSA conjugated 1:16 with phosphorylcholine (PC16-BSA) were purchased from Biosearch Technologies (Novato, CA). NIP14-BSA and PC16-BSA were conjugated to a Cys-containing peptide terminated with a His12 tag (ASTGTASACTSGASSTGSH12) using succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce) following manufacturer’s protocols. ICs were formed by mixing 10 nM His12 tagged NIP14-BSA or PC16-BSA with 20 nM rabbit anti-BSA (for IgG-IC) or F (ab′)2 rabbit anti-BSA [for F(ab′)2-IC]. Recombinant ICAM-1 with a His12 tag was a gift of J. Huppa (Stanford University, Palo Alto, CA). The mouse ICAM-1/huFc chimera protein with a His12 tag was purchased from R&D Systems. Conjugation of His12-tagged NIP14-BSA and PC16-BSA to Cy5 and His12-tagged ICAM-1 to AlexaFluor488 were performed following manufacturer’s protocols (19 (link)).
Cy3- and Cy5-conjugated Fab goat Abs specific for mouse IgM and IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Cy3-conjugated Fab rat mAb specific for mouse IgM (clone no. II/41) was purchased from Rockland (Gilbertsville, PA). Rabbit IgG Abs specific for BSA (rabbit anti-BSA) were purchased from Bethyl Laboratories (Montgomery, TX) and F(ab′)2 rabbit anti-BSA were prepared as described (20 (link)). PE-conjugated and biotin-conjugated rat mAb specific for mouse FcγRIIB (clone no. 2.4G2) and APC-conjugated mouse mAb specific for human FcγRIIB (clone no. FLI8.26) were purchased from BD Pharmingen (San Diego, CA). Biotin-conjugated mouse mAb specific for human FcγRIIB (clone no. AT10) was purchased from AbD Serotec (Raleigh, NC). Biotin-conjugated F(ab′)2 goat Abs specific for mouse IgG and IgM, biotin-conjugated F(ab′)2 rabbit Abs specific for human IgM, and streptavidin were purchased from Jackson ImmunoResearch Laboratories. Rat mAb specific for mouse FcγRIIB (clone no. 190907) was purchased from R&D Systems (Minneapolis, MN) and Fabs mAb 190907 were prepared using an immobilized papain kit (Pierce, Rockford, IL) following the manufacturer’s protocol. Conjugations of Abs with Alexa514, 568, or 647 were performed using Alexa Fluor mAb labeling kits (Molecular Probes, Eugene, OR) following manufacturer’s protocols. BSA conjugated 1:14 with 4-hydroxy-5-iodo-3-nitrophenyl acetyl (NIP) (NIP14-BSA) and BSA conjugated 1:16 with phosphorylcholine (PC16-BSA) were purchased from Biosearch Technologies (Novato, CA). NIP14-BSA and PC16-BSA were conjugated to a Cys-containing peptide terminated with a His12 tag (ASTGTASACTSGASSTGSH12) using succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce) following manufacturer’s protocols. ICs were formed by mixing 10 nM His12 tagged NIP14-BSA or PC16-BSA with 20 nM rabbit anti-BSA (for IgG-IC) or F (ab′)2 rabbit anti-BSA [for F(ab′)2-IC]. Recombinant ICAM-1 with a His12 tag was a gift of J. Huppa (Stanford University, Palo Alto, CA). The mouse ICAM-1/huFc chimera protein with a His12 tag was purchased from R&D Systems. Conjugation of His12-tagged NIP14-BSA and PC16-BSA to Cy5 and His12-tagged ICAM-1 to AlexaFluor488 were performed following manufacturer’s protocols (19 (link)).
