Tube Formation assay Freshly isolated LEC were seeded on fibronectin (50 mg ml21, Roche) coated wells at a density of 105 cells/0.3 ml in m-Slides 8 well (Ibidi) or 104 cells/0.01 ml in m-Slides Angiogenesis (Ibidi) and allowed to adhere overnight. Cells were rinsed with EBM-2 and overlaid with 0.2 ml (or 0.04 ml) of Matrigel diluted 1:1 in ice- cold EBM-2 containing growth factors and/or inhibitors at double the required final concentration. All treatments and controls were adjusted to contain the same final concentration of DMSO (0.2%). Matrigel was allowed to solidify and was further overlaid with EBM-2 containing the indicated treatments. Cells were grown for 24 or 48 h in Matrigel and images were captured using an inverted microscope (Olympus MVX10) and F-view camera (Soft Imaging System) and analysed using CellR software (Olympus Soft Imaging System). Average vessel diameter was quantified using Lymphatic Vessel Analysis Protocol (LVAP) [28] and ImageJ [29] software. The number of vessel branch points was quantified using AngioTool software [30]. Proliferation assay Proliferation assays were performed using the CellTiter 96H AQueous One Solution Cell Proliferation Assay reagent (Pro- mega), as per manufacturer’s instructions. All experiments were performed in 96 well plates with 4–5 replicates. Briefly, freshly isolated LEC were diluted to 16105 cells ml21 in EGM-2MV (Lonza) and 0.1 ml of cells was added to each well. Plates were cultured at 37uC/5% CO2 for 16–18 h and then serum starved for 4 h in control media comprising EBM-2 containing 0.5 mg ml21 Albumax II (Invitrogen). Control and treatment groups were added to cells and incubated for 48 h. Cell proliferation was measured either by trypsinization and cell counting using a haemocytometer, or following the addition of CellTiter 96H AQueous One Solution Cell Proliferation Assay reagent (0.02 ml) to each well and incubation for a further 4 h. Absorbance was measured at 490 nm on a FLUOstar OPTIMA microplate reader (BMG LABTECH). Migration scratch assay All experiments were performed in pre-marked 96 well plates with 4–5 replicates. Freshly isolated LEC were diluted to 56105 cells ml21 and 0.1 ml of cells was added to wells. Cells were cultured in EGM-2MV (Lonza) at 37uC/5% CO2 until confluent. Cells were then starved in EBM-2 containing 0.5% FBS (base media) overnight. A single scratch was made in each confluent cell layer using a 200 ml pipette tip and cells were washed gently in EBM-2 (Lonza). Images at time 0 (initial) were captured using an inverted phase contrast microscope (Olympus CKX41) at 46 magnification. Cells were then incubated in base media (Control) or base media containing FGF2 (10 ng ml21), FGF2 (10 ng ml21)+SU5402 (10 mM) or VEGF-C (200 ng ml21) for 8 h. Images were captured 8 h later using an inverted phase contrast microscope (Olympus CKX41) at 46 magnification. For imaging purposes, wells were completely filled with EBM-2 to overcome distortion caused by the meniscus. Initial and final scratch areas were measured using ImageJ version 1.41 software [29] and the difference between the 0 and 8 h measurements was expressed as migration area. Statisitics For proliferation and migration assays with multiple treatments, randomised complete block design with sub sampling was assumed. Data was analysed using SAS/STATH 9.2 software and the PROC MIXED procedure. If a difference between Lymphangiogenesis Assays Using Primary Mouse LEC treatments was detected at the 5% level, pair-wise comparisons of the means (with Sidak adjustment) were used to determine P values. Unless otherwise stated, error bars in each figure represent s.e.m. of at least three independent experiments with multiple replicates of each treatment. Results Isolation, purity and shot-term culture of primary mouse lymphatic and blood vascular endothelial cells Primary lymphatic (LEC) and blood vascular endothelial cells (BEC) were purified from single cell suspensions of embryonic mouse skin. Cell suspensions were first depleted of hematopoietic cells including macrophages, using a magnetic bead isolation approach coupled with anti-F4/80 and anti-CD45 antibodies. Two rounds of depletion were employed to ensure complete removal of these potentially contaminating cell types. LEC were then isolated using an anti-LYVE-1 antibody and following this, BEC were selected using an anti-CD31 antibody (Figure 1a). The purity of isolated LEC and BEC was assessed immediately following cell isolation and RNA extraction, by real-time RT- PCR for a panel of genes characteristic of LEC, BEC, hematopoietic, vascular smooth muscle and epithelial identity (Figure 1b, c). These assessments revealed that genes characteristic of LEC identity including Lyve1, Prox1, Flt4, Pdpn and Ccl21 were highly enriched in embryonic dermal LEC (Figure 1b), while genes including Flt1, Nrp1 and Cd34 were highly enriched in embryonic dermal BEC (Figure 1c). Pan-endothelial genes including Pecam1 and Kdr (encoding VEGFR-2) were expressed in both LEC and BEC populations, though were higher in BEC (Figure 1b). Both primary embryonic endothelial cell populations were negative for macrophage (Emr1, encoding F4/80), vascular smooth muscle (Acta2, encoding smooth muscle actin, alpha 2) and epithelial (Krt14, encoding keratin 14) genes (Figure 1c), indicating a high degree of specificity in our cell isolation approach. Purified LEC and BEC cultured on fibronectin coated dishes grew rapidly to confluence. Interestingly, the morphology of each cell type was distinct; primary dermal LEC grew in clusters and were large, oak-leaf shaped cells (Figure 2a–c). By comparison, primary dermal BEC were smaller, more spindle shaped, tended to grow in tube-like structures and grew more slowly than LEC (Figure 2d–f). These features mirror the morphology of lymphatic and blood vascular capillaries in embryonic skin; lymphatic capillaries are of a much larger calibre than their blood vascular counterparts (Figure S1a). Immunostaining for proteins charac- teristic of lymphatic and blood vascular capillaries in primary embryonic LEC and BEC following 3 days in culture confirmed the highly pure nature of isolated primary LEC and BEC (Figure 2, Figure S2). LEC were positive for Prox1, VEGFR-3 and CD31 and negative for macrophage (F4/80) and blood vascular markers (CD34) (Figure 2a–c). Interestingly, purified embryonic LEC, like adult mouse LEC [8], exhibited heterogeneous levels of LYVE-1 in culture (Figure 2a). BEC were positive for CD34, VEGFR-2 and Nrp1 and negative for Prox1 and LYVE-1 (Figure 2d–f). Though LEC were successfully passaged up to 4 times, cell growth and proliferation slowed with each passage. In addition, expression levels of some markers of LEC identity including Ccl21 and thrombospondin1 (Thbs1) were dramatically reduced once LEC were cultured. For this reason, all of our experiments were performed with freshly isolated cells in their first passage. Intriguingly, LEC isolated from E16.5 embryos attached and proliferated more readily than LEC isolated from E18.5, perhaps reflecting elevated plasticity at this earlier developmental time- point. Essentially the same cell isolation approach was used to PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40497