2.4 Preparation of single cell suspensions

Single cell suspensions were prepared as described previously . Briefly, spleen and ghLN were mechanically disrupted with a steel strainer. Liver sections were cleared of peripheral blood by perfusion with ice-cold PBS-EDTA before further cell extraction. Samples from lungs and perfused liver were then digested (collagenase IV (2 mg/ml; Biochrom), DNase I (0.1 mg/ml; Sigma-Aldrich)) for 1 h at 37°C in serum-free cell culture media (1:1 Ham’s F12/IMDM). Tissue residuals were removed by short centrifugation. The cells were washed with serum-supplemented cell culture media (10% fetal calf serum) and used for flow cytometry.