Liver dissociation kit

Murine livers were obtained and dissociated using the liver dissociation kit (Miltenyi Biotec) in accordance with the manufacturer's instructions. Dissociated hepatic cells were passed through a 40-µm filter, and red blood cells (RBCs) were lysed by RBC lysis buffer (eBioscience). The cell suspension was washed with PEB buffer (0.5% [v/v] BSA, 5 mM EDTA in PBS). The immune cells were enriched from the cell suspension by gradient centrifugation using Optiprep density gradient medium (Sigma-Aldrich). Briefly, the cells were suspended in 10 mL of 33.3% Optiprep diluted in PEB. Seven milliliters of PEB was carefully layered on top of the cell suspension without disturbing the interface of two layers. The cell suspension was centrifuged at 500g for 20 min at 4°C without brake at the end of centrifugation. Immune cells at the interface between two layers were extracted, washed with PEB, and analyzed by flow cytometry. Cells were also fixed and stored at −80°C for mass cytometry.

According to the experiment design, single-cell suspensions were prepared from desired organs. In brief, the BM single-cell suspension was flushed out of femurs. The harvested spleen was teased apart into a single-cell suspension with a syringe plunger. We prepared single-cell suspensions from the lung and liver with Lung Dissociation Kit (Miltenyi Biotec, Nordrhein-Westfalen, Germany) and Liver Dissociation Kit (Miltenyi Biotec), respectively, per the manufacturer’s instructions. To analyze tumor-infiltrating immune cells, tumor mass was dissociated with a tumor dissociation kit (Miltenyi Biotec) following the manufacturer’s instructions. After obtaining single cells from different organs, erythrocytes were eliminated with red blood cell (RBC) lysis buffer. Single blood cells were generated from directly lysed blood with RBC lysis buffer. For CD115 expression analysis, cold EDTA buffer was used for BM and blood cell preparation^{66 (link)}. Cells were ready for use after passage through a 70-μm nylon cell strainer.

Liver nonparenchymal cells (NPC) were isolated using a liver dissociation kit and the gentle MACS dissociator (both Miltenyi Biotec) as recommended by the manufacturer with some modifications. In brief, after liver dissociation, cells were resuspended in 1 mL cold HBSS buffer (w/o Ca²⁺ and Mg²⁺). The cell suspension was mixed with a double volume of freshly prepared 30% HistoDenZ (Sigma-Aldrich) and overlaid with 1 mL of cold HBSS buffer. After centrifugation (1500× g, 4 °C, w/o break), the cell containing interphase was retrieved and washed. Erythrocytes were lysed using a hypotonic buffer.
Spleen cells were isolated using a 40 µM cell strainer (Greiner Bio-One) to obtain a single-cell suspension. In parallel settings, spleen cells were resuspended in medium (IMDM, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (all from Sigma-Aldrich, Deisenhofen, Germany), and 50 µM ß-mercaptoethanol (Roth, Karlsruhe, Germany)) containing 5% FCS (PAN).
Bone marrow cells (2 × 10⁵/mL) were seeded in 12-well cell cluster plates (Greiner Bio-One) in an IMDM-based culture medium (see above) supplemented with 10 ng/mL recombinant murine GM-CSF (R&D Systems, Wiesbaden, Germany). Culture media was replenished on days 3 and 6 of culture.

Liver mononuclear cells were isolated using the liver dissociation kit provided by Miltenyi Biotec (Bergisch Gladbach, Germany). Briefly, whole liver tissue was placed in a C tube with a pre-warmed dissociation mix and placed on the gentleMACS™ dissociator for homogenization. The sample was incubated at 37°C for 30 minutes under continuous rotation at 100 rpm before being placed back on the gentleMACS™ dissociator. The cell suspension was then filtered and washed with 5 mL Dulbecco’s Modified Eagle Medium (DMEM). Centrifugation at 300xg and room temperature (25°C) was performed for 10 minutes before the supernatant was discarded. The cell pellet was then resuspended into 6 milliliters (mL) of staining buffer (PBS with 2% heat-inactivated FBS and 1 mM EDTA) and transferred to a 15 mL conical tube containing 3 mL of 100% Percoll. The cells were then resuspended in the resulting 33% Percoll solution were placed in the centrifuge at 2000 revolutions per minute (rpm) for 15 minutes at room temperature. After discarding the supernatant, red blood cell lysis was performed for 5 minutes on ice and washed with staining buffer. The resulting single-cell suspension was filtered and counted using a Bio-Rad TC20 Automated Cell Counter (Hercules, CA).

Mice were sacrificed, and the livers were digested with a LIVER dissociation kit (Miltenyi Biotec, Germany) and dissociated into a cell suspension. Then, immune cells were isolated by Ficoll-Hypaque (DAKEWE, SZ, China) density gradient centrifugation from the cell solution. Isolated cells were washed twice in HBSS and resuspended at 2 × 106 cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 50 µM 2-mercaptoethanol.