Isolation and Analysis of Lymphoid and Splenic Cells

LNs, and spleens where indicated, were incubated in digestion medium (RPMI, 0.1 mg/ml DNase I (Invitrogen), 0.1–0.2 mg/ml liberase (Roche) and 0.8 mg/ml dispase (Roche)^{19 (link)}. Collected single-cell suspensions were filtered. Spleen samples underwent red blood cell lysis. For flow cytometry, cells were stained with Ghostdye510 viability dye (Tonbo Biosciences) in PBS, followed by labeling with CD45 (30-F11, Invitrogen), CD45.2 (104, BD Biosciences), PDPN (8.1.1, Biolegend), CD31 (MEC 13.3, BD Biosciences) and Ki67 (SolA15, eBioscience), Madcam-1 (MECA-89, BD), CD21/CD35 (Clone: 7G6, BD), Cpt1a (8F6AE9, Abcam), CD4 (GK1.5, Invitrogen), B220 (RA3–6B2, BD), IL-17RA (PAJ-17R, Invitrogen) and CountBright™ Absolute Counting Beads (Molecular Probes). For detection of apoptosis, FRC were stained with FITC Active Caspase-3 Apoptosis Kit (BD Biosciences), as per manufacturer’s protocol. For in vivo cell cycle analysis, mice were injected i.p. with 1 mg of bromodeoxyuridine (BrdU flow kit; BD Biosciences) 24 h before sacrifice. Data acquired with a FACS Fortessa (BD Biosciences), analyzed using FlowJo (Tree Star).