Mouse lsec binding magnetic beads

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.

Mouse LSECs were isolated by previously described two-step collagenase perfusion technique with modifications.^{15 (link)} In brief, the liver was perfused with Liver Perfusion Medium (Invitrogen), and dissociated by Liver Digest Medium (Invitrogen). The NPCs were fractionated with Percoll (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) gradient centrifugation with 75% stock Percoll solution and 35% stock Percoll solution. LSEC faction was isolated by mouse LSEC-binding magnetic beads (Miltenyi, Auburn, CA, USA) and Dynabeads Magnetic Beads conjugated with anti-mouse CD31 antibody (MEC13.3, BD Biosciences). Expression of Id1, CXCR7, HGF and Wnt2 messenger RNA was determined. Primary human LSECs were procured from ScienCell Research Laboratories (catalog no. 5000, Carlsbad, CA, USA). Expression of factor VIII was validated by immunostaining. Akt-LSECs were derived from isolated LSECs that were transfected with the pCCL. PGK lentiviral vector with mouse constitutively active Akt1 (myristoylated Akt: myrAkt).^{63 (link)} After starving in serum-free medium, 500 000 LSECs were seeded and stimulated with 10 ng ml⁻¹ SDF-1. LSECs were also treated with 30 μm Wortmannin (Sigma-Aldrich).