approaches has limitations; very small numbers of primary cells can be purified from collecting lymphatic vessels, there is considerable heterogeneity in the endothelial cells that line large collecting vessels compared to lymphatic capillaries, and immor- talisation and extended culture alters the molecular properties and identity of LEC. Moreover, while the culture of primary human LEC over several passages has been feasible, the culture of primary mouse LEC has been challenging. We sought to optimise methodology for the isolation and short-term culture of primary embryonic mouse LEC in order to establish lymphangiogenesis assays able to: 1. Measure LEC proliferation, migration and tube formation in response to established and candidate pro- and anti- lymphangiogenic stimuli. 2. Assess gene function in defined aspects of lymphangiogenesis by utilising siRNA to silence gene expression in wild-type LEC. Here, we describe methodology for the isolation, short-term culture and transfection of highly pure populations of primary embryonic mouse LEC and BEC. Furthermore, we have optimised robust assays to quantify primary mouse LEC prolifer- Lymphangiogenesis Assays Using Primary Mouse LEC ation, migration and three-dimensional tube formation in vitro. We have utilised these techniques to investigate the roles of FGF2 and VEGF-C signalling in lymphangiogenesis in vitro and report that FGF2 and VEGF-C drive distinct cellular events; FGF2 potently promotes LEC proliferation while VEGF-C stimulates LEC sprouting and elongation. Our data uncover a previously unrecognised role for signal transduction via FGFR1 in primary mouse LEC and suggest that this signalling axis is likely to play a key role during lymphangiogenesis in vivo. Methods Animal studies and Ethics Statement Experiments using mice were performed using C57Bl/6 mice and were approved and conducted in accordance with the SA Pathology/Central Health Network (CHN) Animal Ethics Com- mittee and Australian National Health and Medical Research Council (NHMRC) guidelines. Figure 1. Isolation and purity of primary mouse embryonic dermal lymphatic (LEC) and blood vascular (BEC) endothelial cells. (a) Schematic representation of mouse embryonic dermal endothelial cell isolation. Skin of E16.5 embryos was removed and digested to generate a single cell suspension. Macrophages and hematopoietic cells were depleted using anti-F4/80 and anti-CD45 antibodies, in combination with anti-rat magnetic beads. LEC were captured using anti-LYVE-1 antibody and anti-rabbit magnetic beads, prior to isolation of BEC using anti-CD31 antibody and anti-rat magnetic beads. (b) Analysis of mRNA levels of established markers of LEC identity in LEC and BEC isolated from E16.5 dermis. (c) Analysis of mRNA levels of known markers of BEC (Flt1, Nrp1, Cd34), macrophage (Emr1), vascular smooth muscle (Acta2) and keratinocyte (Krt14) identity in LEC and BEC isolated from E16.5 dermis. Data were normalised to Actb and show mean 6 s.d. of triplicate samples. Data are representative of at least three independent cell isolations, each prepared from multiple litters of embryos. doi:10.1371/journal.pone.0040497.g001 PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40497