Lymphangiogenesis Assays Using Primary Mouse LEC Figure 3. FGF2 stimulates primary mouse LEC proliferation. (a) Primary LEC were cultured in EBM-2+0.5 mg ml21 Albumax (Control) or EBM- 2+0.5 mg ml21 Albumax containing FGF2 or VEGFC at the indicated concentrations for 48 h. LEC proliferation was measured using the CellTiter 96H AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean 6 s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). (b) FGF2 stimulated LEC proliferation is inhibited by an FGFR tyrosine kinase inhibitor but not by VEGFR inhibitors. Primary LEC were cultured in EBM-2+0.5 mg ml21 Albumax (Control), or EBM- 2+0.5 mg ml21 Albumax and FGF2 (10 ng ml21), together with the tyrosine kinase inhibitors SU5402 (10 mM, FGFR inhibitor), SU5416 (5 mM, VEGFR-2 inhibitor) or MAZ51 (5 mM, VEGFR-3 inhibitor). LEC proliferation was measured using the CellTiter 96H AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean 6 s.e.m. and are derived from 3 independent cell isolations prepared from multiple litters of embryos and 5 replicates of each treatment (n = 15). **P,0.01 ***P,0.001. doi:10.1371/journal.pone.0040497.g003 mouse VEGFR2 (R&D Systems), goat anti-mouse VEGFR3 (R&D Systems), rabbit anti-KDR (Upstate/Millipore), goat anti- rat Neuropilin-1 (R&D Systems), rabbit anti-Flg (FGFR1) (C-15) (Santa Cruz). Preparation of skin cell suspensions Where practicable all procedures were carried out on ice and as quickly as possible. Intact uteri were removed from 2–3 pregnant female mice and placed in ice-cold DMEM (Sigma) until dissection. Skin was removed in large pieces from 15–25 embryos and transferred to ice-cold 10 ml HHF (5% FBS, 10 mM Hepes Buffer in HBBS) until all embryos were processed. Dissected skin was rinsed twice with DHF (DMEM/20%FCS/10 mM Hepes) and replaced with 10 ml DHF containing 25 mg Collagenase Type II, 25 mg Collagenase Type IV and 10 mg Deoxyribonu- clease I (all from Worthington), followed by incubation at 37uC for 30 min, pipetting with a wide-bore transfer pipette every 5 min to assist tissue dissociation. Skin cell suspensions were filtered through a 40 mm cell strainer and rinsed with 2 volumes ice-cold DHF. Filtrates were centrifuged at 300 g for 10 min. Cell pellets were resuspended in 5 ml HHF and cell counts performed. Cell suspensions from adult skin were prepared as follows: ears from 3– 4 adult mice were peeled apart, excess cartilage was removed and remaining skin was cut into small pieces. Tissue digestion was Figure 4. FGF2 and VEGF-C promote migration of primary mouse LEC. (a) Confluent monolayers of primary LEC were scratched and cultured in EBM-2+0.5% FBS (Control), or EBM-2+0.5% FBS containing FGF2 (10 ng ml21) 6 SU5402 (10 mM) or VEGF-C (200 ng ml21) for 8 h. Dotted white lines mark the boundaries of the wound at 0 h. Scale bars represent 125 mm. (b) Quantification of area migrated in 8 h. Data represent mean 6 s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). *P,0.05, **P,0.01, ***P,0.001. doi:10.1371/journal.pone.0040497.g004 PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40497