Isolation and Characterization of Primary Mouse Dermal Lymphatic Endothelial Cells

Primary mouse dermal LECs derived from C57BL/6 embryos were obtained and maintained in complete mouse endothelial cell media with supplements (C57-6064L & M1168, Cell Biologics). All the in vitro cell culture experiments were performed within passage 5. For Dot1l inactivation, LECs were grown in the LEC culture media containing 2 µM EPZ5676 (reconstituted in DMSO, A12735, Adooq) for 7 days. The EPZ5676-treated LECs were subjected to ChIP-Seq analysis. Isolation of LECs from embryonic skin was described in a previous study^{65 (link)}. Briefly, E15.5 embryonic skin was removed and enzymatically dissociated with media containing type II and IV collagenase, and DNaseI (LS004176, LS004188, and LS006344, respectively; Worthington Biochemical Corp.) for 20 min at 37 °C. After filtration through a 40-µm cell strainer, dissociated cells were incubated in both F4/80 and CD45 antibodies (13-4801 and 13-0451, respectively; eBioscience) for 1 h at RT to deplete macrophage and collected using goat anti-rat IgG-coated microbeads (130-048-101, Miltenyi Biotec). The F4/80(–)/CD45(–) cells were incubated with Lyve1 antibody (13-0443, eBioscience) and secondary antibodies. The Lyve1(+) LECs were collected and analyzed by RNA-Seq and qRT-PCR analyses.