Next monoclonal antibodies was indeed purchased of Pharmingen (Hillcrest, Calif
Phone staining and disperse cytometry.
Single-cell suspensions were prepared from different lymphoid organs and incubated for 10 min at 10 6 cells/20 ?l on ice in staining buffer (phosphate-buffered saline [PBS] containing 0.5% bovine serum albumin [BSA] and 0.01% NaNstep 3) with optimal amounts of fluorescein isothiocyanate-, phycoerythrin-, or biotin-conjugated antibodies. ): S7 (anti-CD43), B3B4 (anti-CD23), and Ly1 (anti-CD5). The following antibodies were prepared: RA3-6B2 (anti-B220), R33- (anti-IgM), 1.3-5 (anti-IgD), and Cfo-1 (anti-Thy1.2). Flow cytometric analysis was performed on a FACScan cytometer (Becton Dickinson & Co., Mountain View, Calif.).
Analysis off B-phone expansion and upregulation from activation markers.
Splenic B cells was in fact purified because of the depletion out of non-B cells to the MACs columns (Miltenyi Biotec, Bergisch Gladbach, Germany) having anti-CD43 antibody combined so you can magnetized beans (Miltenyi Biotec) because the discussed previously (17). The brand new love from B tissues was subject to fluorescence-activated cellphone sorter analysis, and the agreements out-of B tissues of 95% purity were utilized. B cells was sparked having goat anti-IgM antibody (2.5 ?g/ml) (Diaburg, Germany), anti-CD40 antibody (0.six ?g/ml) (Pharmingen), and you will IL-cuatro (25 U/ml) (Genzyme Corp., Boston, Mass.). The research off telephone expansion and you will upregulation off activation markers are did because the revealed in earlier times (step three, 14).
Investigation off necessary protein term and you may tyrosine phosphorylation.
For the analysis of protein expression, cells were lysed in lysis buffer (10% glycerol, 1% Triton X-100, 20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 ?g of leupeptin per ml, 10 ?g of aprotinin per ml).