Cd45r b220 microbeads

GC B cells (B220⁺, CD95⁺, CD38^-) were sorted from RagC^mut and RagC^wt splenocytes after 12 days of SRBC immunization in a BD FACSAria Ilu (Becton Dickinson) and InFlux (Cytopeia-Becton Dickinson) cell sorters. Total RNA from sorted GCB was extracted using TRIZOL (Invitrogen, #15596026) and PicoPure RNA Isolation kit (Arcturus, #12204-01) following manufacturer instructions. For gene expression profiling of murine lymphomas, B220⁺ were isolated from mouse lymphoma tumors by immunomagnetic enrichment with CD45R (B220) microbeads (Miltenyi Biotech # 130-049-501).

For polyclonal T cells, cells were isolated from the spleens and lymph nodes of c-Myc-GFP mice via mechanical passage through a Falcon 70 uM cell strainer (Thermo Fisher Scientific) and lysis of red blood cells in hypotonic solution. Single cell suspensions were enriched for T cells via CD45R (B220) microbeads and MACS separation (Miltenyi; Auburn, CA). Enriched cells were then stimulated on plate-bound anti-CD3 (1 ug/mL), anti-CD28 (1 ug/mL) (BioXCell West Lebanon, NH), and recombinant human CD54/ICAM (0.5ug/ml) produced in insect cells for 30–36 hrs¹ . OT-I Tg T cells were similarly isolated and enriched to ~96% purity and were either stimulated as above or overlain onto bone-marrow-derived dendritic cells previously pulsed with 100 nM of SIINFEKL peptide (60193-1, Anaspec Inc.; Freemont, CA) at 37°C for 1 hr. Co-cultures were incubated at 37°C for 36–40 hrs. In all stimulations for microscopy experiments, nocodazole (100 ng/mL) (Sigma-Aldrich) was added to culture for 4 hours and washed off before subsequent imaging. All T cells were cultured in RPMI 1640 (Gibco; Grand Island, NY) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine, 0.05mM 2-mercaptoethanol, 100units/ml penicillin and 100µg/ml streptomycin at 37°C in 5% CO₂.

B220⁺ cells were purified from mouse lymphoma tumors by immunomagnetic enrichment with CD45R(B220) microbeads (Miltenyi Biotech). RNA extraction was performed using TRIzol (Ambion) following the manufacturer’s protocol.

Spleens were excised from Foxp3–GFP reporter mice approximating two months of age. Following erythrocyte lysis, B cells were depleted using CD45R (B220) Microbeads (Miltenyi Biotec, Cat. #: 130-049-501). T_Regs were FACS-sorted based on forward scatter (FSC), side scatter (SSC) and GFP positivity. Cells were washed with 1 × PBS and resuspended at a final concentration of 7.5 × 10⁴ cells per 100 μL. 7.5 × 10⁴ cells were delivered retro-orbitally (r.o.) to 21-day-old C57BL/6 and Snai2/Snai3 cDKO recipients. As a negative control, a group of cDKO animals were left untransferred. Animals were monitored out to 60 days of total lifespan. Bodyweight was measured over the first 4 weeks post-transfer and plasma samples were collected at four weeks post-transfer. At the completion of the each experiment, WT and cDKO animals were euthanized and analyzed via FACS.