Recombinant fgf10

Mono- or co-culture of EpiSPC and rMC was performed as described previously [30 ]. In brief, flow sorted cells were counted, resuspended and mixed with growth factor reduced matrix (BD Biosciences) diluted with EpiSPC medium (α-MEM, 10% FCS, 1x pen/strep, 1x insulin/transferrin/selenium, 2mM L-glutamine, 0.0002% heparin) at a 1:1 ratio. Cell suspensions were seeded in 12mm cell culture inserts (0.4μm pore size, Millipore) in a 24-well plate and incubated for 5 min at 37°C, 5% CO₂ to allow gelling. EpiSPC medium was then added to the bottom wells of the plate. In selected experiments, 50ng/ml recombinant Fgf10 and 30ng/ml recombinant Hgf (both R&D systems) or anti-Fgf10 or control Ab were added to the medium at day 2 of culture. For matrix digestion a preheated dispase/collagenase I (Boehringer, Gibco) mixture (3 mg/ml) was added and a single cell suspension was obtained for re-seeding or single-cell sorting. Images were taken with a DM IL LED microscope and a corresponding camera MC170 HD (Leica).

Recombinant FGF10 (catalogue: 345-FG-025) was purchased from R&D Systems (Minneapolis, MN). Antibodies against FGF10 (ab115825), MAP-2 (ab5392), GFAP (ab7260), tubulin (ab179513) and t-PI3K (ab22653) were purchased from Abcam (Cambridge, MA). Antibodies against p65 nuclear factor-κB (NF-κB, sc-372), IκB (sc-371), TNF-α (sc1351) and IL-6 (sc-1265) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against p-Akt (2965), t-Akt (9272) and p-PI3K (4288) was purchased from Cell Signaling Biotechnology (Danvers, MA). Fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit was purchased from Promega (Medison, WI). Caspase-3, −8 and −9 activity colorimetric kits were purchased from Abcam. 2,3,5-triphenyltetrazolium chloride (TTC), wortmannin and Akt1/2-KI were purchased from Sigma Chemical Company (St. Louis, MO)

LACE assay was performed as previously described [5 (link),25 (link)]. Briefly, frozen sections were incubated in 0.05% NABH₄/PBS for 15 min. After several washes in PBS, sections were incubated in 0.1M glycine at 4°C overnight. Control sections were incubated with Heparitinase I (Seikagaku) before proceeding. Sections were then treated with 1% BSA/TBS solution for 10 min before incubation with 3 μM recombinant Fgf2 (R&D Systems) and 9 μM recombinant human Fgfr1α(IIIc)-Fc (R&D Systems); 20 nM recombinant Fgf10 (R&D Systems) and 20 nM recombinant human Fgfr2β(IIIc)-Fc (R&D Systems); 30nM recombinant Fgf8b (R&D Systems) and 100 nM recombinant human Fgfr3(IIIc) (R&D Systems) at 4°C overnight. Formation of ternary structure was detected by incubation of 1/200 anti-human IgG (Fc-specific) Cy3 (Sigma) in 1% BSA/TBS. n = 5 for wild-type embryos, n = 3 for mutant embryos.