Human basic Fibroblast Growth Factor Recombinant

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Human basic Fibroblast Growth Factor Recombinant

$70.00$700.00


accession P09038


Source Optimized DNA sequence encoding Humanbasic Fibroblast Growth Factormature chain was expressed in Escherichia Coli.
Molecular weight Native humanFGF basic isgenerated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calulated mass of kDa. Recombinant bFGFis a disulfide-linked homodimeric protein consisting of amino acid residue subunits, and migrates as an approximately17 kDa protein under non-reducing and 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 stimulation of thymidine uptake by BaF3 cells expressing FGF receptors, corresponding to a specific activity of ≥2x units/mg.
Protein Sequence MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS
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 Human Basic FGF was lyophilized from a.2 μm filtered solution inM NaCl,20mM PB pH.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 O35082
Interactor O43353
Interactor P21802 FGFR2_HUMAN
Interactor P22607 FGFR3_HUMAN
Interactor Q802A9
Interactor P11362 FGFR1_HUMAN
Interactor P22455 FGFR4_HUMAN
Biological Process Angiogenesis
Biological Process Differentiation
Molecular function Developmental-protein
Molecular function Growth-factor
Molecular function Heparin-binding

Methods

Neural progenitor cell isolation and maintenance

  • All procedures were performed in sterile fashion in a class II biosafety cabinet.
  • A representative portion (2.5 cm) of three regions (thoracic, cervical and lumbar) of the spinal cord was used for Neural Progenitor Cell (NPC) isolation as described previously.
  • Briefly, tissue was diced and a single cell suspension was obtained by enzymatic dissociation of the tissue at 37 °C for approximately 30–40 minutes with 2.5 U/ml papain , 250 U/ml of DNase I , and 1 U/ml neutral protease .
  • After dissociation, the cell suspension was mixed with DMEM/F12 with 10% fetal bovine serum (FBS, , ), passed through a 70 μM filter, and centrifuged.
  • The cell pellet was resuspended in DMEM/F12 with 10% FBS and combined 1:1 with percoll .
  • The cell/percoll mixture was centrifuged at 20,000 g for 30 min at room temperature and the low buoyancy fraction (10 ml) above the red blood cell layer was collected.
  • s were washed and…
  • All procedures were performed in sterile fashion in a class II biosafety cabinet.
  • A representative portion (2.5 cm) of three regions (thoracic, cervical and lumbar) of the spinal cord was used for Neural Progenitor Cell (NPC) isolation as described previously.
  • Briefly, tissue was diced and a single cell suspension was obtained by enzymatic dissociation of the tissue at 37 °C for approximately 30–40 minutes with 2.5 U/ml papain , 250 U/ml of DNase I , and 1 U/ml neutral protease .
  • After dissociation, the cell suspension was mixed with DMEM/F12 with 10% fetal bovine serum (FBS, , ), passed through a 70 μM filter, and centrifuged.
  • The cell pellet was resuspended in DMEM/F12 with 10% FBS and combined 1:1 with percoll .
  • The cell/percoll mixture was centrifuged at 20,000 g for 30 min at room temperature and the low buoyancy fraction (10 ml) above the red blood cell layer was collected.
  • s were washed and resuspended in NPC medium containing DMEM/F12 supplemented with 10% FBS , 10% BIT9500 , 1% N2 supplement , 20 ng/ml of FGF-2 , 20 ng/ml of EGF , and 20 ng/ml of PDGF-AB .
  • NPCs were cultured on fibronectin coated plates and after 24 hours, the media was replaced with serum-free NPC medium.
  • Half of the medium was subsequently replaced every 2 days.
  • Cells were passed when 60–70% confluence was reached in about 3–4 weeks.

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Cell dissociation and culture

  • Enzymatic digestion was used to dissociate human CSCs from atrial appendages as described previously 2+/Mg2+-free PBS buffer.
  • Thereafter, the samples were minced into small pieces (∼1 mm) and washed extensively in fresh cold PBS buffer.
  • The chopped tissues were then transferred into 50 ml tube and incubated with collagenase II (30 U/ml) in 37°C shaker for 60 min.
  • The loosened tissues were further mechanically dissociated by gently pipetting.
  • The undigested tissue clumps were allowed to settle down by gravity on ice for 10 min followed by transferring the supernatant into another 15 ml tube.
  • After centrifuge at 1,400 rpm for 5 min, the cell pellet was resuspended in fresh CSC culture medium consisting of Ham's F12 , 10% FBS , 10 ng/ml human bFGF , 0.2 mM L-glutathione, and 0.005 U/ml human erythropoietin (both from, ).
  • The cells suspension was then plated in culture flask and cultured in 37°C incubator supplied with 95% air/5% CO2.
  • Next day,…
  • Enzymatic digestion was used to dissociate human CSCs from atrial appendages as described previously 2+/Mg2+-free PBS buffer.
  • Thereafter, the samples were minced into small pieces (∼1 mm) and washed extensively in fresh cold PBS buffer.
  • The chopped tissues were then transferred into 50 ml tube and incubated with collagenase II (30 U/ml) in 37°C shaker for 60 min.
  • The loosened tissues were further mechanically dissociated by gently pipetting.
  • The undigested tissue clumps were allowed to settle down by gravity on ice for 10 min followed by transferring the supernatant into another 15 ml tube.
  • After centrifuge at 1,400 rpm for 5 min, the cell pellet was resuspended in fresh CSC culture medium consisting of Ham's F12 , 10% FBS , 10 ng/ml human bFGF , 0.2 mM L-glutathione, and 0.005 U/ml human erythropoietin (both from, ).
  • The cells suspension was then plated in culture flask and cultured in 37°C incubator supplied with 95% air/5% CO2.
  • Next day, the medium was entirely changed to remove all dead cells and cell debris.
  • The adherent cells were continuously cultured with medium change every other day or split at 4–5 days with TrypLE Express .
  • The subsequent analyses were done on the entire un-fractional population of cultured cells or after c-kit MACS and/or Lin-dep.

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Materials

  • Fetal bovine serum (FBS) Medium (DMEM), Penicillin–Streptomycin , HEPES and trypsin/EDTA were obtained from .
  • Ascorbic acid-2-phosphate, dexamethasone, sodium-β-glycerophosphate fibrinogen and thrombin were obtained from .
  • 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 .
  • All other substances were of analytical or pharmaceutical grade and obtained from.

Cell culture

  • GM97, GM1600, and GM1605 were established from surgically resected specimens from glioblastoma patients at the University of in accordance with protocols approved by the University of as previously described 2 cell culture incubator .
  • Glioblastoma and medulloblastoma cells grown as sphere cultures were collected by gentle centrifugation (800×g, 5 min) and trypsinized with 0.05% TrypLESelect for 5 min by pipetting.
  • Cells were washed twice with phosphate buffered saline (PBS), counted, and seeded at a density of 2000 cells per 200 µL into 96-well ultralow-attachment plates (Corning).

MN Induction of Human Mesenchymal Stem Cells

  • Subconfluent hMSCs were incubated in growth medium with 1 mM β-mercaptoethanol for 24 hours and were subsequently treated with 2 mM β-mercaptoethanol.
  • After 3 hours, cultures were transferred to growth medium containing 1 µM retinoic acid (RA) and 5 µM forskolin (FSK).
  • At day 3, the cultures were maintained in growth medium supplemented with 1 µM RA, 5 µM FSK and 10 ng/ml recombinant human basic-fibroblast growth factor (bFGF, , ).
  • After an additional 4–6 days of culture, with media changes every 2 days, the cells were induced in the presence of 1 µMA, 5 µM FSK, 10 ng/ml bFGF and 200 ng/ml recombinant human sonic hedgehog (SHH , , , ).