Lymphangiogenesis Assays Using Primary Mouse LEC Figure 5. FGF2 and VEGF-C promote tube formation of primary mouse LEC. (a) Primary LEC were cultured for 24 h and imaged immediately following the addition of Matrigel. (b) Primary LEC were cultured for 24 h followed by addition of Matrigel alone or Matrigel containing FGF2 (10 ng ml21), VEGF-C (200 ng ml21) or a combination of FGF2 and VEGF-C. Images were captured after a further 48 hours. (c) Primary LEC were cultured for 24 h followed by addition of Matrigel containing FGF2 (10 ng ml21) or a combination of FGF2 (10 ng ml21) and VEGF-C (200 ng ml21) and tyrosine kinase inhibitors SU5402 (10 mM, FGFR1), SU5416 (5 mM, VEGFR-2) or MAZ51 (5 mM, VEGFR-3). Three replicates of each treatment were performed and images are representative of at least three independent cell isolations. Inset panels in (c) illustrate magnified views of boxed regions. Scale bars represent 250 mm. Quantification of average vessel diameter (d) using Lymphatic Vessel Analysis Protocol (LVAP) [28] and ImageJ [29] software and branch points per well (e) using AngioTool software [30], for each treatment indicated. Data show mean 6 s.e.m. and are derived from 2 independent cell isolations, each prepared from multiple litters of embryos, and 3 replicates of each treatment (n = 6). *P,0.05, **P,0.01, ***P,0.001. doi:10.1371/journal.pone.0040497.g005 carried out at 37uC for 60 min with frequent pipetting and single cell suspensions were processed as described above. Cells were harvested 48 h after the second transfection and subjected to RNA and protein analyses. Depletion of F4/80(+)/CD45(+) haematopoietic cells Crude dermal cell mixtures were centrifuged at 400 g for 5 min and pellets resuspended at approximately 108 cells ml21 in HHF together with anti-F4/80 and anti-CD45 antibodies (1:100). Cells were mixed by gentle rotation at 4uC for 5 min, washed with 10– 20 volumes MACS Buffer (PBS, 2 mM EDTA, 5% FBS), centrifuged as above and resuspended at 108 cells ml21 in MACS Buffer. F4/80(+)/CD45(+) cells were depleted using goat anti-rat IgG Microbeads (MiltenyiBiotec), using 200 ml beads per 108 cells, according to manufacturer’s instructions. F4/80(2)/CD45(2) negative cells were collected and the entire process repeated to ensure maximal depletion of contaminating haematopoietic cells. Isolation of Lyve1(+) LEC and CD31(+) BEC F480(2)/CD45(2) cells were centrifuged at 400 g for 5 min and resuspended at approximately 108 cells ml21 in HHF with anti-Lyve1 antibody (1:200). Lyve1(+) LEC were purified using goat anti-rabbit IgG Microbeads according to manufacturer’s instructions and eluted cells reapplied promptly to a fresh MACS MS column to increase purity. F480(2)/CD45(2)/Lyve1(2) cells were centrifuged at 400 g for 5 min and resuspended at approximately 108 cells ml21 in HHF with anti-CD31 antibody (1:100). CD31(+) BEC were purified using goat anti-rat IgG Microbeads according to manufacturer’s instructions and eluted cells reapplied to a fresh MACS MS column. Isolated primary dermal LEC and BEC were either used immediately for RNA and protein isolation, or plated on fibronectin (50 mg ml21, Roche) coated dishes in EBM-2 medium supplemented with EGM-2MV SingleQuots (Lonza) and grown at 37uC in 5% CO2. Approximate yields: 20 embryos2–46107 total cells (approx 0.6% LEC, 2% BEC) 20 embryos1–26108 total cells (approx 1% LEC, 4% BEC) 5 embryos1–26108 total cells (approx 1% LEC, 4% BEC) Transfection with siRNA LEC transfection with siRNA was performed using Lipofecta- mineTM 2000 (Invitrogen) according to manufacturer’s instruc- tions. The sequence of the siRNA used to target Fgfr1 (NM_010206.2, 59-GAAGACUGCUGGAGUUAAUTT-39) was designed and synthesized by Shanghai GenePharma Co., td. Briefly, freshly isolated LEC were seeded on fibronectin (50 mg ml21, Roche) coated 24-well dishes or m-Slides 8 well (Ibidi) at a density of 0.5–16105 cells/well, or on fibronectin coated 96-well plates at 26105 cells/well. Cells in 24-well dishes were cultured overnight and transfected with 20 pmol siRNA (33 nM final concentration). After a further 24 h, media was replaced and cells were subjected to a second transfection using 40 pmol siRNA (66 nM). Cells in 96-well plates were transfected in the same manner, using 4 and 8 pmol siRNA, respectively. Immunostaining For analysis of cell purity, primary LEC and BEC isolated from E16.5 embryos were grown on fibronectin (50 mg ml21, Roche) coated m-Slides 8 well (Ibidi) for 3 days, fixed with 4% phosphate- buffered paraformaldehyde (PFA) and incubated with primary antibodies overnight at 4uC as previously described [26]. Transfected LEC were fixed 48 h after the second transfection. Alexa FluorH -488, -555 and 647 conjugated secondary antibodies (Invitrogen) were used for visualization. Cells were mounted in Prolong Gold with DAPI (Invitrogen). Images were captured at room temperature using a Bio-Rad Radiance 2100 confocal microscope (Bio-Rad Laboratories) equipped with 3 lasers (488 nm Argon ion, 543 nm Green HeNe and 637 nm Red Diode) attached to an Olympus IX70 inverted microscope (Olympus). Adobe Photoshop CS5 version 12.0 (Adobe) was used for subsequent image processing. RNA Analysis Total RNA was isolated from transfected cells using TRIzolH reagent (Invitrogen) according to the manufacturer’s instructions. For investigation of mRNA expression, total RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitro- gen) with a mixture of oligo dT and random hexamer primers. Primers used for real-time RT-PCR analysis are shown in Table S1. PCR was performed with RT2 Real-Time SYBR Green/Rox PCR master mix (SA Biosciences) and analysed on a Corbett Rotor-Gene 6000 Real-Time PCR machine. Data were normal- ised to the housekeeping gene Actb as previously described [27]. Western Blotting Transfected cells were either lysed using T-PER reagent including Protease Inhibitors (Thermo Scientific) or following RNA extraction using TRIzolH reagent, whereby protein was recovered from the remaining organic phase according to the manufacturer’s instructions. For analysis of protein levels in transfected cells, equal volumes of protein lysate isolated from three separate transfections were pooled and electrophoresed on 8% SDS-PAGE gels prior to being transferred to PVDF (Perkin Elmer). Western blots were performed according to standard protocols using anti-Flg (C-15) (Santa Cruz) and anti-b-actin (Sigma) antibodies, followed by anti-rabbit AP (GE Healthcare) and anti-mouse Cy5 (GE Healthcare). Signal was detected using ECF Western Blot substrate (GE Healthcare) and blots were directly scanned on a Typhoon Imager (GE Healthcare). Densitometry was performed with ImageQuant TL software (GE Healthcare). PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40497