Cd19 biotin

Spleen, MLN and kidney were collected and mashed in 70-μm cell strainers with C10 media (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, all from Life Technologies, Grand Island, NY). For splenocytes, red blood cells were lysed with RBC lysis buffer (eBioscience, San Diego, CA). To isolate intestinal epithelial cells (IECs), the entire intestine including small intestine and colon was opened longitudinally and cut into pieces. The pieces were incubated twice in EDTA-DTT solution and intensively vortexed to harvest IEC-enriched fractions. For surface marker staining, cells were blocked with anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with Attune NxT flow cytometer (Thermo Scientific). For intracellular staining, Foxp3 Fixation/Permeabilization kit (eBioscience) was used. Anti-mouse antibodies used in this study include: CD3-APC-eFluor 780, CD8-PE-Cy7, CD4-PerCP-Cy5.5, RORγT-PE, CD3e-biotin (eBioscience); CD45-APC-Cy7, IL-10-BV421, IL-17A-APC, CD49b-biotin, CD19-biotin (Biolegend, San Diego, CA); Biotin-FITC (MACS). Flow cytometry data were analyzed with FlowJo.

For the isolation of cells in the CNS, mice were anesthetized and transcardially perfused with PBS. CNS tissues were cut and digested in DMEM with Collagenase A (1 mg/ml, Roche) and DNase I (0.1 mg/ml, Roche) at 37 °C for one hour. Cells were homogenized using a 70-μm cell strainer (BD Biosciences) before leukocytes were separated using 30 and 70% Percoll gradients and subsequently stained for 30 min in FACS buffer (PBS/2.5%FCS/10 mM EDTA/ 0.01% NaN3) with the following primary antibodies: CD45-PacificBlue (Biolegend, 103126), CD19-biotin (Biolegend, 115503), CD3E-APC (Biolegend, 100311). Oligonucleotide barcodes conjugated to phycoerythrin (PE) and streptavidin (TotalSeq™-C0951 PE Streptavidin, TotalSeq™-C0952 PE Streptavidin and TotalSeq™-C0953) were added to biotinylated anti-CD19 antibodies to maintain sample identity following pooling of B cells for each experimental condition. CD19 + B cells were sorted individually for each mouse using a BD LSRFortessa (BD Biosciences) and BD FACSDiva (BD Biosciences, v8.0.2) using appropriate filter sets and compensation controls. B cells arising from the same experimental condition were subsequently pooled and used as input to single-cell immune repertoire sequencing. Data were analyzed using FlowJo software (v10).