Human Fibroblast Growth Factor 10 Recombinant

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Human Fibroblast Growth Factor 10 Recombinant

$70.00 – $2,700.00

SKU: RKO15520 Categories: FGF family, FGF family Tags: fgf-10, FGF10, O15520

Description

Methods (52)


accession	O15520

Source	Optimized DNA sequence encoding Human Fibroblast Growth Factor-10 mature chain was expressed in Escherichia Coli.
Molecular weight	Native human FGF10 is generated by the proteolytic removal of the signal peptide and propeptide, this molecule has a calculated molecular mass of approximately 19 kDa. Recombinant Fibroblast Growth Factor is a monomer protein consisting of 170 amino acid residue subunits,and migrates as an approximately 19 kDa protein reducing conditions in SDS-PAGE.
Purity	>96%, as determined by SDS-PAGE and HPLC
Biological Activity	The ED(50) was determined by the dose-dependent proliferation of BaF3 cells expressing FGF receptors, and was found to be <0.5ng/ml. , corresponding to a specific activity of.0 x Units/mg.
Protein Sequence	QAL GQDMVSPEAT NSSSSSFSSP SSAGRHVRSY NHLQGDVRWR KLFSFTKYFL KIEKNGKVSG TKKENCPYSI LEITSVEIGV VAVKAINSNY YLAMNKKGKL YGSKEFNNDC KLKERIEENG YNTYASFNWQ HNGRQMYVAL NGKGAPRRGQ KTRRKNTSAH FLPMVVHS
Endotoxin	Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation	RecombinantFGF10 was lyophilized from.2 μm filtered PBS solution, pH7.0 .
Reconstitution	A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage	The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage	This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.

Interactor	P21802 FGFR2_HUMAN
Interactor	P21803 FGFR2_MOUSE
Molecular function	Growth-factor

Methods


Endothelial Sprouting Assay Endothelial sprouting assay was performed as described ⁴ ESC were seeded in 35 mm dishes in 1.2 mg/ml collagen, type I neutralized with 0.1 N NaOH. Collagen media contained 15% FBS, 450 µm MTG, 10 µg/ml insulin , 50 ng/ml human vascular endothelial growth factor (VEGF, , ), 100 ng/ml human basic fibroblast growth factor (FGF-2, , ) in IMDM. Cells were incubated at 37°C, 5% CO₂. At day 6, 200 µl media without collagen was added and EB were analyzed on day 11. Image analysis was performed using AxioVision LE Software .
Materials Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), Penicillin–Streptomycin (Pen–Strep), HEPES and trypsin/EDTA were obtained from . Ascorbic acid-2-phosphate, dexamethasone, sodium-β-glycerophosphate, Triton X-100, fibrinogen and thrombin were obtained from (St. Louis, ). Basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP2) were obtained from . Endothelial Growth Medium-2 (EGM) was obtained from . Phosphate buffered saline (PBS) and proteinase K were obtained from Fisher Scientific . All other substances were of analytical or pharmaceutical grade and obtained from Sigma-Aldrich.
Cell culture Human CAPs were cultured at a density of 6000 cells/cm² in medium consisting of equal amounts of IMDM (PAA)/DMEM/Ham's F12 medium containing 5% human serum, 1% penicillin/streptomycin, 20 ng/ml basic fibroblast growth factor and 10 ng/ml epithelial growth factor . Human cardiac fibroblasts were cultured in Lung/Cardiac Fibroblasts Basal Medium (Cell Applications, Inc. San Diego, USA) plus supplements (CELL Applications). CAR and CD55 expression flow cytometry analysis was performed on CAPs and cardiac fibroblasts of the same passage number. Murine HL-1 cells were cultured in Claycomb medium (SAFC Biosciences, Kansas, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 100 µM norepinephrine and 2 mM glutamine. Chinese hamster ovary (CHO) cells and CHO cells expressing human CAR (a kind gift of J.M. Bergelson, Children's Hospital of Philadelphia, Philadelphia) were cultured in Hams F12 Human CAPs were cultured at a density of 6000 cells/cm² in medium consisting of equal amounts of IMDM (PAA)/DMEM/Ham's F12 medium containing 5% human serum, 1% penicillin/streptomycin, 20 ng/ml basic fibroblast growth factor and 10 ng/ml epithelial growth factor . Human cardiac fibroblasts were cultured in Lung/Cardiac Fibroblasts Basal Medium (Cell Applications, Inc. San Diego, USA) plus supplements (CELL Applications). CAR and CD55 expression flow cytometry analysis was performed on CAPs and cardiac fibroblasts of the same passage number. Murine HL-1 cells were cultured in Claycomb medium (SAFC Biosciences, Kansas, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 100 µM norepinephrine and 2 mM glutamine. Chinese hamster ovary (CHO) cells and CHO cells expressing human CAR (a kind gift of J.M. Bergelson, Children's Hospital of Philadelphia, Philadelphia) were cultured in Hams F12 medium with 10% FBS and 1% penicillin/streptomycin. The CAR antibody-producing hybridoma cell line RMCB (kindly provided by M. Anders, Department of Interdisciplinary Endoscopy, University Hospital Hamburg Eppendorf, Germany) was cultured in suspension in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin. Read more
Human mesenchymal stem cells (MSCs) isolation Human bone marrow (BM) was aspirated from the iliac crest of healthy donors. Fresh BM was cultured in flasks seeding 10 µl BM cells/cm² with medium'>alpha-minimum medium'>essential medium (α-MEM) supplemented with 2 mM L-glutamine, 15% fetal bovine serum (FBS) , 100 U/ml Penicillin, 0.1 mg/ml Streptomycin and 1 ng/ml of fibroblast growth factor-basic (FGF-b , , ) ³ cells/cm² and cultured under the same conditions.
Cell culture and sphere-forming assay The sphere forming assay was performed as described previously by . with minor modifications [
HaCaT Cells HaCaT cells, an immortalized human keratinocyte line (kind gift of Dr P. Boukamp; ₂. Cells were passaged weekly in T75 flasks and all the experiments were performed with sub-confluent cells. HaCaT cells at 70% confluence were stimulated with hFGF7, hFGF10 or hFGF22 at a concentration of 100 ng/ml, co-treated with(300 ng/ml). FGF dependent stimulation was blocked by 30 minute pre-treatment with the FGFR specific inhibitor, PD173074 (1.7 µM).
MSCs were placed into step one medium, which consists of DMEM supplemented with SPN, 2mM L-glutamine, 20ng/ml human epidermal growth factor (hEGF, R&D Systems, Minneapolis, MN, ), 20ng/ml human basic fibroblast growth factor (hbFGF, R&D Systems) and 10μL/ml N2 supplement (insulin 5μg/ml, progesterone 20nM, putrescin 100μM, selenium 30nM, transferrin 100μg/ml, , , ). 72 hours later, the MSCs were placed into step two medium consisting of DMEM supplemented with SPN, 2mM L-glutamine, 1mM dibutyryl cyclic AMP (dbcAMP), 0.5mM isobutylmethylxanthine (, from), 20 ng/ml hbFGF, 50ng/ml human neuregulin1-β1 and 5 ng/ml Platelet-Derived Growth Factor-AA (PDGF). MSCs grown in medium'>serum-free medium containing DMEM, glutamine and SPN served as untreated controls. Due to species-specific toxicity, when inducing mouse MSCs the concentrations of IBMX and cAMP were halved in the step two medium.
Cell culture, staining, and imaging Satellite cells were identified by FACS and single cells were deposited into 96-well plates in myogenic medium: DMEM/F12 medium without l-glutamine (Cell Gro, Manassas, VA; 15-090-CV) containing 20% FBS , 10% horse serum (26050-088), 50 ng/μL human basic fibroblast growth factor (, , 100-18), 1% penicillin/streptomycin (15140-122), 1% Glutamax , and 0.5% chick embryonic extract (US , , ; ).
Cell culture The human retinoblastoma cell line, WERI-Rb1, obtained from the American Type Culture Collection , was maintained in RPMI-1640 Medium with 10% fetal bovine serum (FBS, , ) [₂ and observed under inverted microscopy every other day.
Generation and administration of UC-MSCs UCs (n = 10, clinically normal pregnancies, approved by the Qilu hospital’s human research ethics committee) were excised and washed in a 0.1 mol/l phosphate buffer (pH 7.4) to remove excess blood. The cords were dissected and the blood vessels were removed. The remaining tissues were cut into small pieces (1–2 mm³) and placed in plates with low-glucose medium'>ulbecco-modified medium'>Eagle medium (L-MEM) , supplemented with 10% fetal bovine serum (FBS), 2 ng/mL vascular endothelial growth factor (VEGF& , , ), 2 ng/mL epidermal growth factor (EGF& ), 2 ng/mL fibroblast growth factor (FGF& ), 100 U/ml penicillin, and 100 μg/ml streptomycin . Cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂. The media were changed every 3–4 d. Adherent cells proliferated from individual explanted tissues 7–12 d after initiating incubation. At this time, the small tissue pieces were removed from the culture and the adherent fibroblast-like… UCs (n = 10, clinically normal pregnancies, approved by the Qilu hospital’s human research ethics committee) were excised and washed in a 0.1 mol/l phosphate buffer (pH 7.4) to remove excess blood. The cords were dissected and the blood vessels were removed. The remaining tissues were cut into small pieces (1–2 mm³) and placed in plates with low-glucose medium'>ulbecco-modified medium'>Eagle medium (L-MEM) , supplemented with 10% fetal bovine serum (FBS), 2 ng/mL vascular endothelial growth factor (VEGF& , , ), 2 ng/mL epidermal growth factor (EGF& ), 2 ng/mL fibroblast growth factor (FGF& ), 100 U/ml penicillin, and 100 μg/ml streptomycin . Cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂. The media were changed every 3–4 d. Adherent cells proliferated from individual explanted tissues 7–12 d after initiating incubation. At this time, the small tissue pieces were removed from the culture and the adherent fibroblast-like cells were cultured to confluence, which subsequently took 2–3 weeks in culture. The cells were then trypsinized using 0.25% trypsin (Gibco-BRL) and passaged at 1 × 10⁴ cells/cm² in the medium described above. The cells were used after five or more passages. Read more