Isolation and Purification of Liver and Spleen Leukocytes

The spleens and livers were flushed by heart perfusion with PBS. Single-cell suspension of splenocytes was prepared as described before (38 (link)). Liver leukocytes were either isolated using the mouse liver dissociation Kit and the gentleMACS dissociator device (both from Miltenyi Biotec) according to the manufacturer’s instructions or, alternatively, minced with scissors, suspended in 3 ml of digestion medium (IMDM-complete with 0.02 mg L⁻¹ of Collagenase D and 0.01 mg L⁻¹ of DNAse; both from Sigma-Aldrich), transferred to Falcon tubes, and incubated (30 min, 37°C) while the suspension was sheared frequently. After 30 min, 2 ml of fresh digestion medium was added, and the livers were incubated for an additional 15 min. Afterwards, 60 μl of 0.5 mol L⁻¹ ethylenediaminetetraacetic acid (EDTA) was added, and samples were rested (5 min, 37°C) to stop enzymatic digestion. Cell suspension was passed through 100-μm cell strainers, washed with PBS, and afterwards passed through a 70-μm cell strainer, followed by centrifugation (300 rcf, 10 min) at room temperature (RT). Erythrocyte lysis was performed as described previously (38 (link)). The resulting pellet was resuspended in 10 ml of 35% EasyColl/PBS and centrifuged (1,800 rpm, 10 min, RT, no brake). Leukocyte pellet was resuspended in IMDM-complete and stored on ice.