2.5. Preparation of splenocytes

Dissected spleens were trimmed from fat and connective tissue and single cell suspensions were prepared by forcing the passage of splenic tissue through a fine metallic mesh back into fresh cell culture medium (RPMI 1640 with GlutaMAX; Thermo Fisher Scientific) with 10% heat inactivated fetal bovine serum (Sigma Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma Aldrich). Cell clusters were disrupted by pipetting and the neat suspension was transferred to new tubes. The cells were washed by decanting the supernatant after centrifugation (10 minutes, 200 g), and subsequent resuspension in fresh medium. Splenocytes were isolated from the cell suspension by density gradient centrifugation with Lymphoprep™ (Stemcell Technologies, Vancouver BC, Canada) according to the manufacturer’s instructions. The isolated cells were washed again, counted in a hemacytometer, and used for analysis immediately.