Lsriii flow cytometer

For intracellular cytokine staining, cells were incubated for 3 h with 100 ng/mL of PMA and 1 μM of ionomycin (all from Sigma-Aldrich, Saint Louis, MO, USA), brefeldin A, and monensin (all from eBioscience, San Diego, CA, USA). After washing cells with cold PBS containing 1.5% FBS, cells were stained with APC-Cy7-conjugated anti-CD45.2 mAb and PE/Cy7-conjugated anti-CD4 mAb (eBioscience, San Diego, CA, USA) for surface staining. Cells were then washed and stained with PerCp-Cy5.5-conjugated anti-IFNγ mAb, APC-conjugated anti-IL-17 mAb (all from BioLegend, San Diego, CA, USA) and PE-conjugated anti-RORγt mAb (eBioscience, San Diego, CA, USA) after incubation with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for 30 min at 4 °C. Cells were analyzed by LSR III flow cytometer (BD Bioscience, San Jose, CA, USA). Data were analyzed with FlowJo (TreeStar, Ashland, OR, USA).

A total cell number of 10⁶ single cells from each (left and right) pln was stained using APC-conjugated anti-mouse CD4 (Clone GK1.5, BioLegend, San Diego, USA), BV510-conjugated anti-mouse CD8a (clone 53-6.7, BioLegend), PerCPCy 5.5-conjugated anti-mouse CD45R/B220 (clone RA3-6B2, BioLegend), PE/Cy7-conjugated anti-mouse CD185/CXCR5 (clone L138D7, BioLegend), and BV421-conjugated anti-mouse CD279/PD-1 (clone 29.F1A12, BioLegend). Samples were analyzed on a BD Biosciences LSRIII flow cytometer and Tfh were identified as CD4/CXCR5/PD-1 co-expressing cells (Meli and King, 2015 (link)). 5 × 10⁴ Tfh were sorted and the repertoire of TCRβ clonotypes was identified as described above (Supplementary file 3).

Approximately 2 × 10⁵ cells per sample were stained with the indicated antibodies. Non-specific staining was blocked using buffer containing 10% BSA, human IgG (Sigma) and normal mouse serum (Sigma). Then, the cells were stained using directly conjugated isotype or antigen-specific antibodies. Live cells were gated using FSC/SSC characteristics and exclusion of 7-AAD (BD Biosciences). The phenotype was evaluated on an LSRIII flow cytometer (BD Biosciences) using DIVA software (BD Biosciences). Absolute cell counts were determined by CountBright bead assay (Invitrogen). Day 6 in vitro generated plasmablasts were labelled with anti-CD38 and 7-AAD prior to sorting on a BD Biosciences Influx.