F ab 2 fragment of anti mouse igm

Splenic B cells were purified by negative selection using CD43 beads (Miltenyi Biotec). Purified B cells were cultured in media, 10 μg/ml F(ab′)₂ fragment of anti-mouse IgM (Jackson ImmunoResearch), 10 μg/ml LPS, 5 μg/ml CpG, or 10 μg/ml Pam3CSK4 (InvivoGen) at 37 °C for the indicated times. In some experiments, the cells were pre-treated with different concentrations of the inhibitors U0126, LY294002 (Cell Signaling Technology), NSC668394, SB203580 (Calbiochem), or PS1145 (Sigma-Aldrich) for 1 hour before stimulating with LPS. Lysates were prepared and immunoblotting performed as described (9 (link), 11 (link)). All immunoblotting antibodies were from Cell Signaling Technology, except for IκB, actin (Santa Cruz Biotechnology, Inc.) and ezrin (EMD Millipore). PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) were used to stimulate B cells ex vivo 1.5 h after in vivo LPS injection. siRNAs to ezrin, MyD88, TRIF, IRF3, and control siRNAs (Dharmacon) were used at 2 μM according to manufacturer’s instructions and as described previously (14 (link)).

Splenic B and T cells were purified by negative selection using CD43 beads or mouse pan T cell isolation kit II (Miltenyi Biotecc), respectively. Purified B cells were stimulated with 10 μg/ml F(ab′)₂ fragment of anti-mouse IgM (Jackson ImmunoResearch Laboratories) for the indicated times. B and T cell lysates were prepared and immunoblotting performed as described (21 (link)). All immunoblotting antibodies were from Cell Signaling Technology, except for actin (Santa Cruz Biotechnology), Igα (Abcam), phosphotyrosine (pY) and ezrin (EMD Millipore). To measure intracellular-free calcium levels, purified splenic B cells were loaded with Fluo-3 AM (Molecular Probes) at 37°C for 20 min. Cells were washed, resuspended in DMEM supplemented with 1% BSA (Sigma) and 20 mM HEPES (Sigma), warmed to 37°C for 5 min, and analyzed by flow cytometry. After the baseline was established for 30–40 sec, cells were stimulated with 10 μg/ml F(ab′)₂ fragment of anti-mouse IgM for the indicated duration.