Isolation of Immune Cells from Various Tissues

For isolation of peripheral blood mononuclear cells (PBMC), whole blood was separated by density gradient centrifugation using Pancoll (PAN-Biotech, Germany). PBMC were collected and washed with PBS-EDTA (1 mM; used for all analyses). Cell count was determined using Neubauer improved haemocytometer. Single cell suspensions from spleen and lymph nodes were prepared by mechanically disrupting tissue with a sieve. Lymphocytes from liver were isolated following a modified protocol previously described (45 (link)). In brief, liver samples were perfused with ice-cold PBS-EDTA. Perfused regions were minced with sterile scissors, resuspended in PBS-EDTA supplemented with 100 μM CaCl₂, and digested with Collagenase D (1 mg/ml; Sigma-Aldrich) for 40 min at 37°C. Remaining tissue was removed by short centrifugation. Cell pellet was resuspended in PBS-EDTA and used for flow cytometry. Lymphocytes from lung tissue were isolated by mincing non-perfused lung tissue, followed by enzymatic digestion as described for liver samples. Lung tissue was additionally mashed through a cell strainer with the plunger of a syringe after digestion. Unless otherwise stated, cells were cultured in Ham's F12/IMDM (1:1), supplemented with 10% fetal calf serum (FCS), 2-mercaptoethanol (50 μM), 100 U/ml penicillin, and 100 μg/ml streptomycin.