M 2 me

Splenocytes and MLNs were isolated and red blood cells were lysed using a buffer containing 155 mM NH4Cl, 12 mM NaHCO3 and 0.1 mM EDTA. Cells were resuspended in RPMI 1640 medium containing 10% FBS (Thermo-Fisher Scientific, Rockford, IL, USA), gentamycin, 2 mM L-glutamine, and 5×10⁻⁵ M 2-ME (Invitrogen, Carlsbad, CA, USA).
Cells were cultured with OVA 500 μg/mL for 72h to collect supernatants. Cytokines were measured using BD OptEIA (BD Biosciences, San Diego, CA) mouse ELISA Sets for IL-2, IL-4, IL-5, IL-10, IL-17 and IFN-γ following the manufacturer’s instructions. The limits of detection were 12.5 pg/ml IL-2, 31.25 pg/ml IL-4, 62.5 pg/ml IL-5, 31.3 pg/ml IL-10, 31.3 IL-17, and 25 pg/ml IFN-γ.

Splenocytes were isolated as above and magnetic activated cell sorting (MACS) was used to deplete CD43-expressing cells using LS columns (Miltenyi). Sorted B cells were plated at 2 × 10⁻⁵ cells/well in complete RPMI 1640 media (Invitrogen Life Technologies) containing 10% FBS, glutamine, gentamicin, and 2 × 10⁻⁵ M 2-ME (Invitrogen Life Technologies) in 96-well, flat-bottom plates (Corning). Stimuli include anti-IgM F(ab’)₂ (Jackson ImmunoResearch Laboratories) and anti-CD40 (HM40-3; BD Biosciences) and were added on day 0. Cells were pulsed with 1 µCi of [³H]thymidine (NEN) per well on day 2 and were harvested on day 3 using a semi-automated cell harvester (Skatron), at which point scintillation counting was used to measure [³H]Thymidine incorporation. The average of triplicate wells ± SD is reported.