*2.6. Isolation and Cultivation of LSECs*

The protocol for magnetic-activated cell isolation of LSECs was adapted from [31]. After induction of anesthesia, a cannula was inserted into the portal vein for perfusion with HBSS containing collagenase type 1, leading to removal of blood and dissociation of liver cells from connective tissue. Afterwards, livers were removed, diced, and incubated in the collagenase solution for 20 min for further digestion. The cell suspension was filtered through 100 μm cell strainers and centrifuged several times for removal of collagen and cell debris. To clear the cell suspension of CD45-positive immune cells, the cell suspension was preincubated with anti-CD45 magnetic beads for 15 min at 4 ◦C and transferred onto LD columns in the QuadroMACS separator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The flow-through was centrifuged for 10 min at 500× *g* and 4 ◦C. The supernatant was discarded and the pellet was resuspended in ice-cold MACS-buffer, according to the manufacturer's protocol. For positive selection of LSECs the cell suspension was incubated with anti-CD146 MicroBeads (Miltenyi Biotec). Then the cell suspension was put on cell columns in the OctoMACS separator (Miltenyi Biotec), according to protocol provided by the company. The flow-through was discarded and the columns

were flushed with MACS-buffer containing 0.05% BSA after removal of columns from the magnetic field. The cell suspension was centrifuged and the pellet was resuspended in cell culture medium (DMEM, supplemented with 10% FCS and 1% antibiotics). Cells were cultured in cell culture dishes covered with collagen type I with the following densities: 24-well: 800,000–900,000 cells/well; and 6-well: 3–3.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/well. Culture medium was removed and cells were washed in PBS after 24 h in culture. Cells were treated with serum-free medium containing DMSO, 10 μM TGR5 agonist, 25 μM taurolithocholic acid, or 10 μM forskolin for 24 h.
