70 m filter

To detect intracellular cytokines and chemokines by flow cytometry, mice administered with Proleukin^®/IL-2 and saline-treated control groups of the same experiment were injected with 0.25 mg of BFA (Sigma-Aldrich, USA) at the endpoint of the experiment (144 h) and sacrificed 6 h later. Submandibular blood collection was carried out in EDTA tubes (Greiner Bio-One, Austria), and red blood cells (RBCs) were lysed using RBC lysis buffer (Life Technologies, USA) prior to flow cytometry staining. Spleen and lymph nodes were digested in a mixture of collagenase IV (GIBCO, UK), DNase I (Sigma Aldrich, USA) and meshed through a 70 µm filter (Thermo Fisher Scientific, USA) in DMEM medium (Thermo Fisher Scientific, USA). When necessary, cell suspensions were subjected to red blood cell lysis (GIBCO, UK). The single-cell suspension was washed and re-suspended in media supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA).

Liver samples were collected from the healthy margin of patients undergoing tissue resection for metastases of colorectal cancer or hepatocellular carcinoma. Tissue was dissociated by grinding through a 70 µm filter (ThermoFisher). Duodenal samples were collected by biopsy during endoscopy for routine clinical indications. Duodenal biopsies were incubated for 1 h shaking at 37°C in a solution of R10 + 1 mg/ml Collagenase D (Sigma-Aldrich) + 100 µg/ml DNase I (ThermoFisher). Biopsies were then dissociated by vigorous agitation using a GentleMACS Dissociator (Miltenyi Biotec) and strained through a 70 µm filter. From this point, liver and duodenal samples were processed in the same way. Samples were washed once in R10 media. Mononuclear cells were isolated on a discontinuous 70–35% Percoll gradient (GE Healthcare) by centrifugation at 700 g for 20 min without brake. The interface containing mononuclear cells was collected and washed in R10. Residual red blood cells were lysed using 1× ACK lysis solution and cells were washed two additional times with R10. Tissue-derived cells were used immediately for subsequent experiments.