Isolation and Preparation of Immune Cells

LNs were cut into small pieces and digested with 1 mg/mL collagenase D (Roche), 1 mg/mL dispase II (Roche), and 0.1 mg/mL DNase I (Roche) in cell culture medium containing 2% fetal calf serum (FCS) for 1 h at 37 °C under agitation. Spleens were digested in 2 mg/mL collagenase D (Roche), 0.8 mg/mL dispase II (Roche), and 0.1 mg/mL DNase I (Roche). Otherwise, spleens were weighed and directly grinded in a glass putter with PBS. Samples were filtered through 40- to 70-μm cell strainer to exclude aggregates. If required, the red blood cells were lysed with ammonium-chloride-potassium buffer. The single-cell suspension was washed and prepared in PBS containing 10% FCS and 2.5 mM ethylenediaminetetraacetic acid. Splenic stromal cells were prepared as previously described (29 (link)).