Low tox guinea pig complement

CARTg transgenic mice were generated as described [34] (link)–[36] (link). C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were housed in specific pathogen free facilities at the University of Chicago. Splenic CARTg CD4⁺ T cells were purified by negative selection using magnetic beads and antibody cocktails (Stem Cell Technologies, Vancouver, Canada). T cell depletion of splenocytes used as antigen-presenting cells was performed by labeling T cells with anti-Thy-1 antibody (AT83A hybridoma supernatant) followed by depletion with Low-Tox guinea pig complement (Cedarlane Laboratories, Hornby, Ontario, Canada) and Ficoll-Hypaque centrifugation. All cultures were performed in complete medium consisting of Dulbecco's Complete Eagle Medium ((DMEM) Gibco (Invitrogen), Carlsbad, CA) supplemented with 10% fetal calf serum (Sigma Aldrich, St. Louis, MO), and additives as described [37] (link).

Splenocytes were pooled from N = 2–4 mice for each biological replicate. These cells were first depleted of CD3 cells by complement-mediated lysis in the following way. Splenocytes were incubated with anti-CD3 antibodies (145-2C11, homemade) at a concentration of 0.4 µg/10⁶ cells for 20 min on ice. Cells were then washed and resuspended in media containing reconstituted Low-Tox Guinea Pig Complement (Cedarlane) at a 1:20 dilution and incubated for 30 min at 37 °C. Afterwards, the cells were washed twice with media and then incubated with a rat anti-mouse FcγRII/III antibody (2.4G2; homemade) in FACS buffer for 15 min on ice to prevent subsequent unspecific binding of staining antibodies. Cells were then stained with fluorochrome-labeled antibodies. Stained cells were sorted as single or dual-κ CD3^–CD21⁺CD1d^lowCD24^low FO B cells or CD3^–CD21^highCD1d^high MZ B cells (as shown in Supplementary Fig. 2a). Sorting purity was generally >95% for FO B cells and >70% for MZ B cells in three independent sorting experiments, and numbers of sorted cells varied from 5 × 10⁴ to 8 × 10⁶ depending on the population (5 × 10⁴–16 × 10⁴ for dual-κ cells). Cell sorting was performed using an ICyte Synergy cell sorter (Sony).