Recombinant fgf7

Embryonic lungs used for in vitro experiments were obtained either from genetically modified embryos generated as described above, or from C57BL/6 wild-type embryos.
Embryonic lungs were dissected and cultured on 13 mm Whitman Track-Etch polycarbonate membranes, with 8.0 μm pores (Merck) positioned atop DMEM culture medium in a 24-well culture dish [medium contained: Dulbecco's Modified Eagle Medium (1x DMEM), supplemented with D-Glucose, L-Glutamine, HEPES, Pyruvate, and Phenol red (Gibco), 10% fetal bovine serum (FBS), 1% penicillin (10,000 units/ml)-streptomycin (10 mg/ml)]. Lungs were incubated at 5% CO₂ and 37°C for ~45 min to allow them to settle. At the desired time, recombinant FGF7 (50 ng/ml), FGF10 (250 ng/ml), soluble FGFR2b (5 μg/ml) (R and D Systems), or anti-FGF10 blocking antibody (20 μg/ml) (Santa Cruz Biotechnology) was added to the experimental lungs (as described in Sakaue et al., 2002 (link)), while the vehicle was added to control samples. Lungs were incubated at 5% CO₂ and 37°C for the duration of the experiment.

FTOC was performed as described previously65 (link). Briefly, thymic lobes were isolated from embryos 15.5 days postcoitus and were cultured for 5 days on the top of Nucleopore filters (Whatmann) placed in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM L -glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mM 2-mercaptoethanol containing 1.35 mM 2’-deoxyguanosine (2-dGUO) (Sigma-Aldrich). To test the effect of IL-6, FGF7 and FSP1 using FTOC, 2-dGUO treated fetal thymic lobes were cultured in DMEM plus 10% FBS with recombinant IL-6 (100 ng/ml; Perprotech), recombinant FGF7 (100 ng/ml; R&D) or FSP1 (5 μg/ml). Six days after the stimulation, the lobes were harvested for flow cytometric analysis. For RTOC, in vitro cultured TECs and freshly isolated thymocytes were reaggregated with or without MitoC treated thymic fibroblasts at a ratio of 1:6:1 by centrifugation and the cell pellet of the aggregates were drawn in 2 μl into plastic tips to place onto the surface of Nucleopore filters for 5 days of culture.

Recombinant FGF7 was obtained from R&D Systems, Minneapolis, MN, USA. The metalloprotease inhibitor DPC333 was a gift from Dr. Carl P. Blobel (Weill Cornell Medicine, Graduate School of Medical Sciences, NY, USA) and diluted in DMSO to the indicated concentrations.