Cd45 antibodies

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.

Immunophenotyping was conducted by flow cytometry with immunostaining by monoclonal antibodies against CD 45 as a negative marker and CD 105 as a positive marker for MSC. Flow cytometry was used to assess the immune profile of MSCs, using the standard for MSC as described by the International Society for Cellular Therapy (ISCT). Cells (P2-3) were pelleted and resuspended in 1% bovine serum albumin in in phosphate-buffered saline (BSA in PBS) and counted. Each population containing 10⁵ cells was used for flow cytometry. Cells were stained directly using a concentration of 1:200 of phycoerythrin (PE)-conjugated CD105 antibodies (eBioscience, Germany) and CD 45 antibodies (eBioscience, Germany), and cells were incubated for 1 h. An appropriate isotype-matched control antibody named mouse immunoglobulin G1 (IgG1) K Iso control (eBioscience, Germany) was used in all analyses. The evaluation of the cells was performed using Cell Quest Software (Becton Dickinson, UK) on FACS flow cytometry [14 (link)].