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.