Easycoll separating solution

For analyses of immune cell infiltration, the tumors were processed with a tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Afterwards, the cells were centrifuged with easycoll separating solution (Biochrom) to discard dead cells. The cells were then stained for 30 min at 4 °C with the following fluorescence labeled antibodies: CD4-PCC5.5 (BD Pharmingen), CD8-PE (BD Pharmingen), CD3-V450 (BD Pharmingen), CD11c-PE (BD Pharmingen) and CD45.2-PCC5.5 (eBioscience). Determination of Tregs was performed with FoxP3 Staining Buffer Set and the antibodies CD4-Vioblue, CD25-AF488 and FoxP3-APC (Miltenyi Biotec). Multicolor flow cytometry was performed with the Gallios Flow Cytometer (Beckman Coulter Inc.).

To obtain hepatic lymphocyte suspensions, livers were perfused with PBS, minced through a 100 µm cell mesh using medium containing collagenase D (0.1 U/ml)/DNase (0.1 mg/ml) and digested for 20 min at 37 °C. Subsequently, cells were centrifuged for 3 min at 300 rpm, followed by hepatic lymphocytes purification by density gradient centrifugation (Easycoll separating solution, ρ = 1.124 g/ml, Biochrom AG). Splenic single cell suspensions were prepared as described earlier^{41 (link)}. Briefly, spleens were minced through a 100 µm cell mesh and the resulting single cell suspensions were subjected to erythrocyte lysis.