Isolation of lymphatic endothelial cell population.

<p>FACS was performed on cells isolated from the back skin of wild type mice. (<b>A</b>) Events are first gated on FSC and SSC for debris. The gated events were then plotted to determine Calcein Am incorporation, which will occur only in live cells. We observed a variation in levels of Calcein incorporation in different cell type as shown by the multiple populations observed. We then gated the positive events for caicein Am as shown in the left plot. The right plot shows the viable cells plotted for expression of CD31 and podoplanin, which we identified to be LEC population. (<b>B</b>) Western blotting for PROX-1 and LYVE-1 on cell lysate from isolated CD45^-/CD31⁺/ podoplanin⁺ population and cultured human microvascular lymphatic endothelial cell (HMVEC) shows comparable expression of these lymphatic endothelial markers in both cell types. (<b>C</b>) Isolated CD45^-/CD31⁺/ podoplanin⁺ cells were cultured and stained for podoplanin and PROX-1. Immunofluorecscent image of the cells is shown. Scale bar 100µm. (<b>D</b>) Isolated CD45^-/CD31⁺/ podoplanin⁺ cells and CD45^-/CD31^-/ podoplanin^- were seeded on Matrigel for 24hours and only CD45^-/CD31⁺/ podoplanin⁺ cells were able to form tube like structures. Scale bar 100µm.