Mouse liver dissociation kit

Briefly, tissues were washed twice using PBS supplied with 10% BSA (Sigma, USA), minced into small pieces with the size of 2–4 mm³, and then dissociated using a Human Tumor Dissociation kit (Miltenyi Biotech, Germany). The only exception was the liver biopsy sample which was dissociated with the Mouse Liver Dissociation kit (Miltenyi Biotech, Germany) based on the cell viability results of preliminary experiment. The cell suspension was further filtered through 70 μm SmarterStrainers (Miltenyi Biotech, Germany). Red blood cells were removed using a Red Blood Cell Lysis Solution (10×) (Miltenyi Biotech, Germany). Dead cells were eliminated using a Dead Cell Removal Kit (Miltenyi Biotech, Germany) to increase the efficiency of sorting robust, and the live cells were washed twice, re‐suspended and then used for single‐cell experiments.

Fresh liver tissues were dissociated using a gentleMACS Dissociator (Miltenyi Biotec) and mouse Liver Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach Germany). After separation and purification from the cell suspension by Percoll gradient centrifugation, liver immune cells were stained with antibodies and then subjected to a CytoFLEX LX cytometer (Beckman Coulter, CA, USA). The antibodies used in flow cytometry are listed in Table S1.

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.

For mass cytometry experiment, we used 6‐month‐old (“young”) and 24‐month‐old (“old”) INK‐ATTAC mice. Liver tissue (1.6 g) was dissociated using mouse liver dissociation kit (Miltenyi, #130‐105‐807) according to the manufacturer’s instruction, including cell debris removal step (Miltenyi, #130‐109‐398). Intrahepatic leukocytes were purified by Percoll (Sigma‐Aldrich, #P1644) gradient centrifugation and stained as previously described in detail (Guo et al,2019). CyTOF (Fluidigm) mass cytometry and data analysis were performed by the Immune Monitoring Core at Mayo Clinic as previously described (Guo et al,2019). Visualization of different immune cell clusters was mapped by using a tSNE map. Relative marker intensity and cluster abundance for each sample were plotted using a heatmap. The CyTOF antibody panel used is listed in Appendix Table S1.