Stromal Cell Enrichment from Lymph Nodes

For stromal cell preparation, lymph nodes were torn into small pieces and collected in RPMI 1640 medium containing 2% FCS, 20 mM HEPES pH 7.2 (Lonza), 0.375 mg/ml Collagenase P (Roche) and 25 μg/ml DNaseI (Applichem). Dissociated tissue was incubated at 37 °C for 60 min, with resuspension and collection of the supernatant every 15 min. Following enzymatic digestion, cell suspensions were filtered and washed with PBS containing 0.5% FCS and 10 mM EDTA. For cell-sorting experiments, stromal cells were enriched by depleting CD45⁺ hematopoietic cells and TER119⁺ erythrocytes using MACS microbeads (Miltenyi) as described previously^{7 (link)}. Cell suspensions were directly used for staining with antibodies. For isolation of hematopoietic cells, lymph nodes were gently smashed across a 26-gauge wire mesh and washed with PBS. Cell suspensions were directly used for staining with antibodies or for ELISPOT assays.