The endothelial-specific Dot1l conditional knockout mouse has been described previously (Yoo et al., 2020 (link)). Briefly, E15.5 Tie2-Cre(+); Dot1l2f/2f (cKO), and Tie2-Cre(−); Dot1l2f/2f (Cont) embryo skins were harvested and dissociated in media containing type II and IV collagenase and DNase I (LS006333; Worthington Biochemical Corp.) for 20 min at 37°C. Cells that passed through 40-µm cell strainers were incubated with anti-CD45 and anti-F4/80 antibodies (13–0451 and 13–4801, eBioscience) for 1 h at room temperature to deplete macrophages. F4/80 (−)/CD45 (−) cells were collected and incubated with Lyve1 antibody (13–0443, eBioscience) to collect LECs. Together with the LECs, isolated F4/80 (−)/CD45 (−)/Lyve1 (−) BECs were subjected to RT-qPCR or RNA-seq analysis. All animal studies were reviewed and approved by the Institute of Animal Care and Use Committee (IACUC) of Konkuk University (IACUC#KU18027).
Ls006333
Manufactured by Worthington
The LS006333 is a laboratory equipment product. It is designed to perform a core function within a laboratory setting, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation. Therefore, the description for this product is not available.
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2 protocols using ls006333
1
Isolating Endothelial Cells from Dot1l Knockout Mice
2
Enzymatic Dissociation of Tissue Samples
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Hank’s Buffered Salt Solution (HBSS; H8264–1L, Sigma-Aldrich, St. Louis, MO) supplemented with 5.5 mM glucose (G7021–100G, Sigma-Aldrich), 5.7 mM cysteine (C562–25, Fisher Scientific, Hampton, NH), and 40 U/ml papain (LS003119, Worthington Biochemical, Lakewood, NJ) was titrated to pH 6.5 using potassium hydroxide (KOH) and then incubated for 30 min at 37 °C. After incubation, 10 mM HEPES buffer (SKU# 15,630-106, Invitrogen, Carlsbad, CA) was added to the solution, and it was titrated to pH 7.4 using KOH. About 120 U/ml DNase I (LS006333, Worthington Biochemical), 10 U/ml superoxide dismutase (LS003540, Worthington Biochemical), 10–25 U/ml catalase (C1345–1G, Sigma-Aldrich), and 0.02 mM D-alpha-tocopherol acetate (T1157–1G, Sigma-Aldrich) were added before the solution was filtered using a 0.22 µm pored sterile filter.
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