Quantifying Peripheral B Cells After SpA Challenge

To quantify the effect of SpA infusions on peripheral B cells, spleens were harvested 72 h post-SpA challenge and cell suspensions prepared, as described previously (43 (link), 46 (link), 56 (link)). Cells were maintained on ice throughout. For each mouse, 2 × 10⁶ cells were stained per panel in the presence of 20 μg/ml of Fc block (Pharmingen, Becton Dickinson) followed by staining with: B220-APC (eBioscience), CD3-PerCP Cy5.5 (BD), IgM^a-PE (BD) or IgM^b-PE (BD). For live/dead cell discrimination, we used fixable blue (Invitrogen) following the manufacturer's instructions. After staining, cells were washed and fixed in 4% paraformaldehyde. At least 5 × 10⁵ events per sample were collected on a LSR II flow cytometer (BD) running FACSDiva software. Data were analyzed with FlowJo (Treestar).