Isolation and Culture of Mouse Liver Sinusoidal Endothelial Cells

LSECs were isolated from male C57BL/6 J mice via protocols adapted from modified method^{14 (link),15 (link)}. Briefly, after mice anaesthetized by pelltobarbitalum natricum, the liver was perfused in situ with two steps of Hanks buffer and collagenase solution, respectively, and then excised and digested in perfusion buffer. The resulting supernatant was centrifuged at 50 g for 3 min to eliminate hepatocytes. The resting supernatant enriched in HSCs, KCs and LSECs was separated by Optiprep^TM density gradient medium. The cell fraction between the 8.2 and 17.6% Optiprep^TM enriched in LSECs and KCs was further separated by mouse LSEC binding magnetic beads (Miltenyi, Germany). Purity and viability of isolated LSECs were confirmed by CD146 + F4/80- and 7-AAD + flow cytometry, respectively, and quality of LSECs was detected by fluorescently labeled Ac-LDL.
Human LSEC line SK-HEP1 was purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in 24-well plates with DMEM (Hyclone, South Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) and antibiotics.