Extended Data Fig. 8: Gating strategies used for sorting and characterization of lymphatic and haemogenic endothelial cells. | Nature

Extended Data Fig. 8: Gating strategies used for sorting and characterization of lymphatic and haemogenic endothelial cells.

From: A Prox1 enhancer represses haematopoiesis in the lymphatic vasculature

Extended Data Fig. 8

a, Dorso-anterior regions of E14.5 embryos from wildtype (+/+) or homozygous mutant (Δ/Δ) litters were dissected as indicated, taking care to remove liver, lungs hearts and thymus. 6–8 torsos from a single litter were pooled and digested to generate a single cell suspension. Following F480+ Lin+ depletion, FACS sorted CD45-LYVE1+VECAD+ cells were plated on OP9 feeder layers with cytokines for 7 days. In the case of transcriptomic analyses (Extended Data Figs. 5a,b, 10a), half of the cells from each genotype were processed for RNA (pre-OP9) while the other half were grown on OP9 and then sorted to purify LYVE1+VECAD+ cells (post-OP9). For methylcellulose colony assays all CD45+ cells were FACS purified from OP9 co-cultures, plated into Methocult and cultured for 9–14 days (Fig. 4a). Colonies were enumerated and harvested for FACS analysis (Fig. 4b and Extended Data Fig. 9a). b, FACS analysis of colonies arising after 14 days demonstrates differences and overlap between enhancer mutant and wildtype LEC derived colonies. Venn diagram shows percentages of CD45+ cells also positive for CD41, CD16/32 and cKIT in each genotype. Data are representative of 5 independent experiments. c, Methylcellulose colonies derived from wildtype and enhancer mutant LECs have replating capacity. Colonies were harvested 14 days after initial plating, replated in Methocult™ and assessed at d8. Cells from enhancer mutant colonies demonstrate enhanced proliferation. Data are representative of three independent experiments. Scale bars, 200 µm.

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