Anti cd40 ic10

Purified splenic B cells were incubated for 72 hours at 37^oC in media alone (RPMI 1640 + 10 % FBS + L-glut + pen/strep + β-ME), 5 ug/ml LPS (Sigma) + 50 ng/ml IL-4 (R&D Systems), or 10 ug/ml anti-CD40 (IC10, Invitrogen) + 50 ng/ml IL-4 (R&D Systems). Cells were then stained with antibodies against B220, CD138, and IgG1 and analyzed by flow cytometry (see above).

Purified splenic B cells were stimulated at 37^oC for varying times from 1 minute to 1 hour with media alone (RPMI 1640 + 10 % FBS + L-glut + pen/strep + β-ME), 10 ug/ml anti-IgM F(ab)’2 fragments (Jackson Immunoresearch), 10 ug/ml anti-CD40 (IC10, Invitrogen), 10 ug/ml anti-IgM F(ab)’2 fragments plus 10 ug/ml anti-CD40, or 0.1, 0.3, or 1 ug/ml CD40L (R&D Systems). Cells were lysed in 2x Laemmli sample buffer (Biorad) and equal cell equivalents subjected to SDS page using a 4–15% gradient gel (Biorad). Gels were transferred to nitrocellulose (GE Healthcare) and blocked in 5% milk. Blots were probed overnight at 4^oC with rabbit monoclonal antibodies against pAkt S473 (Cell Signaling Technology), pS6 (Cell Signaling Technology), and β-actin (Cell Signaling Technology) and subsequently for 2 hrs at room temperature with goat anti-rabbit HRP (Biorad). Blots were washed three times in TBST after each antibody incubation. Bands were detected with Clarity ECL reagent (Biorad) and imaged and quantified with a Chemidoc Imaging system (Biorad) and Image Lab software (Biorad).