Anti igm fab2 fragments

Splenocytes were resuspended at 15 × 10⁶ cells/ml in RPMI medium containing 1% FCS, 10 mM HEPES, 1 mM MgCl₂, 1 mM EGTA, and penicillin-streptomycin 1% and 1 μM Indo-1 (Invitrogen). Cells were incubated in a 37°C incubator for 30 minutes. Following incubation, a 5-fold volume of the same buffer (without Indo-1) was added and the cells were centrifuged (300 g, 5 minutes). PBMCs were stained with anti-mouse B220 and anti-mouse CD5, for 30 min and washed 2×. Cells were stored on ice in calcium flux running buffer (HBSS, 1% FCS, 1mM CaCl₂) and an aliquot (0.5 ml; 1 × 10⁶ cells) was warmed (37°C, 5 minutes) prior to initiating calcium flux measurements. During flow cytometry acquisition and analysis B220⁺CD5⁻ cells were gated to establish the B-cell population. Cells were stimulated with liposomes or anti-IgM F(ab)’₂ fragments (Jackson ImmunoResearch) and Indo-1 fluorescence (violet vs. blue) was monitored by flow cytometry (500–1000 events/s) for 3–5 minutes at 37°C. Stimulation always took place 10 seconds after starting acquisition so that background could be established. For experiments involving GFP⁺ vs GFP⁻ mice, the GFP is expressed in all tissues and is not cell type specific, the splenocytes were mixed at a 1:1 ratio prior to loading with Indo-1. Data were analyzed using FlowJo using the kinetics functions.

Splenic B cells were purified by negative selection using anti-CD43 magnetic beads (Miltenyi, Bergisch Gladbach, Germany) according to manufacturer’s protocol. Cells were stimulated at 1×10⁶ cells/ml in 96-well plates with 50 μg/ml LPS (Sigma Aldrich, St. Louis, MO), 10 μg/ml polyclonal anti-IgM, F(ab′)₂ fragments (Jackson ImmunoResearch, West Grove, PA), or 10 μg/ml anti-CD40 (HM40-3) (BioLegend) + 100 U/ml IL-4 (Peprotech, Rocky Hill, NJ). After 72 hours, expansion was measured by flow cytometry using AccuCount 5.27 μm Blank Particles (Spherotech, Lake Forest, IL) according to manufacturer’s protocol.