Isolation and Characterization of Murine Fibroblastic Reticular Cells

FRC cell lines were established from peripheral LNs by long-term culturing as described previously^{74 (link)} with minor modifications. In brief, LNs from 8-week-old C57BL/6 N mice were dissected and disrupted using two 25 G needles before enzymatic digestion with DMEM medium containing 3.5 mg/ml Collagenase D and 40 μg/ml DNase I at 37 °C for 30 min with agitation⁷⁵ . The mixture was then filtered through a 70 μm cell strainer and centrifuged at 300 g for 5 min at 4 °C. The cell pellet was resuspended and cultured in DMEM medium (supplemented with 10% FBS and 1% Penicillin/Streptomycin) (5% CO₂, 37 °C). After 24 h, non-adherent cells were removed, and fresh medium was added to continue culturing until cells reached confluence. Adherent-stromal cells were then trypsinized, and triple-stained with antibodies to identify FRCs: Cd45 Pacific blue (1:100), Cd31 PE (1:100) and gp38 APC (1:100). FRCs were sort-purified using a MoFlo Optical Bench Sorter (Beckman Coulter) to achieve a purity of ≥95%. The sorted cells were immediately cultured in DMEM medium for expansion, and then seeded into 60 mm dishes in a density of 3 × 10⁵ per well to grow until confluence, followed by starvation for overnight and treatment with 10 μM isoproterenol (Sigma-Aldrich), 5 μM forskolin (Sigma-Aldrich), and 5 μM PKA inhibitor H89 dihydrochloride hydrate (Sigma-Aldrich) for 8 h. Culture media were collected to determine the concentration of IL-33 using a mouse IL-33 immunoassay kit (Immunodiagnostics Limited) or LDH using a CyQUANT LDH Cytotoxicity fluorescent assay kit (Thermo Fisher Scientific).