Nycodenz

To isolate non-parenchymal cells (NPC), livers were perfused in situ with digestion medium containing collagenase IV (Sigma-Aldrich, Steinheim, Germany) injected into the portal vein, excised and further incubated in the digestion medium. To eliminate parenchymal cells, the single-cell suspension was subjected to a one-step density gradient centrifugation with 26% Nycodenz (Progen Biotechnik, Heidelberg, Germany). LSEC were isolated from NPC by magnetic cell sorting using anti-CD146 antibody (ME-9F1; BioLegend, Fell, Germany) as previously described [19 (link)]. LSEC adhered over night and were subsequently washed in order to remove non-adherent cells resulting in a purity of higher than 99% [20 (link)]. For transmigration assays, CD4+ T cells were isolated from spleen and lymph nodes using anti-CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) to a purity of at least 95%. Untouched CD4+ T cells used for homing assays were isolated by CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction to a purity of at least 95%.

Thirty L4 stage animals were picked to 60 x 10 cm plates per strain. Plates were confluent with mixed stage animals after four days growth at 20 °C. Progranulin cleavage was observed after starving animals for an additional seventy-two hours at 20 °C. A lysosomal fraction was isolated from a light mitochondrial-lysosomal fraction as previously described [68 (link)] with the following modifications. Animals were collected in 0.25 M sucrose (pH 7.2) and washed twice with 0.25 M sucrose. Lysosomes and mitochondria were separated using a discontinuous Nycodenz (Progen Biotechnik, Germany, #1002424) density gradient. Lysosomes were collected from the 19.8% / sucrose interface and the 26.3 / 19.8% interface and pooled. Lysosomes were diluted five times with 0.25 M sucrose, and pelleted at 37,000 × g for 15 minutes. Cytosolic, ER and lysosomal fractions were confirmed by immunoblotting for specific subcellular fraction markers (LAMP-1, HSC-70, calnexin).

For isolation of KC, HSC, and hepatocytes 20-week-old female or male mice were used. Briefly, mice were anesthetized by ketamine/xylazine injection and perfused in situ through the inferior vena cava with sequential Pronase E (0.4 mg/mL) and Collagenase D (0.8 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) solutions. Liver was removed and digested in vitro with Collagenase D (0.5 mg/mL), Pronase E (0.5 mg/mL) and DNAse I (0.02 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). After 20 min, tissue was filtered through a 70 µm mesh and hepatocytes were separated by centrifugation. Hepatocytes were washed and seeded into collagen-coated plastic tissue culture dish. The remainder of the cells were separated using a Nycodenz (PROGEN Biotechnik, Heidelberg, Germany) gradient centrifugation. HSC or KC were then washed and seeded separately onto plastic tissue dishes in DMEM or RPMI containing fetal serum and incubated at 37 °C with CO₂ overnight. Next morning, medium was changed, and cells were washed and stimulated with CA, DCA, or LCA (100 µM) for 3 h followed by either Nigericin (10 µM) or ATP (5 mM) for 1 h.

A part of each stool sample was submitted to a density gradient in order to separate the microbiota from the rest of the fecal material, according to the method of Courtois and colleagues with some modifications25 (link). Two grams of feces were homogenized in 18 mL of sterile NaCl 0.9% (w/v), in a laboratory paddle blender (Stomacher Lab Blender 400, Seward Ltd. UK) for 1 min. A solution of Nycodenz® 80% (w/v) (PROGEN Biotechnik GmbH, Heidelberg, Denmark) was prepared in ultrapure water, and sterilized at 121 °C for 15 min. A volume of 10.5 mL of the diluted, homogenized fecal sample was placed on top of 3.5 mL of the Nycodenz® solution, and centrifuged for 40 min at 4 °C (10,000 × g, TST41.14 rotor, Kontron, Milan, Italy). The upper phase, containing soluble debris, was discarded after the centrifugation step, and the layers corresponding to the microbiota extracted with 10.5 mL of PBS (Fig. 1) were collected. Cellular suspensions were kept on ice for 5 minutes, in order to allow non-soluble debris to precipitate, were then washed twice, and stored in aliquots of 1 mL, at −80 °C, until DNA extraction was performed. In all the cases, DNA directly from homogenized stool samples, or from the corresponding separated microbiota fractions was extracted using the QIAamp DNA Stool Mini kit (Qiagen Ltd., Strasse, Germany), as described in a previous work26 (link).

30% and 80% Nycodenz (Progen) stock solutions were prepared with HP150 buffer. 20 µl 80% (w/w) Nycodenz was added and thoroughly mixed with 20 µl proteoliposomes. Next 40 µl of 30% (w/w) Nycodenz were overlaid and finally 40 µl HP150 buffer was added as top layer. Samples are spun at 275,000 × g with the S55S swinging bucket rotor (Hitachi) for 60 min at 4 °C. After centrifugation, 20 µl aliquots are taken from the top of the gradient and analyzed with SDS-PAGE and Western blotting.