Mouse basic Fibroblast Growth Factor Recombinant

/, FGF family, Recombinant Mouse Cytokines/Mouse basic Fibroblast Growth Factor Recombinant

Mouse basic Fibroblast Growth Factor Recombinant

$70.00$1,300.00


accession P15655


Source Optimized DNA sequence encoding Mouse basic Fibroblast Growth Factor mature chain was expressed in Escherichia Coli.
Molecular weight Native Mouse FGF basic is generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated mass of 16 kDa. Recombinant mouse bFGF is a monomer protein consisting of 145 amino acid residue subunits, and migrates as an approximately 16 kDa protein under non-reducing and reducing conditions in SDS-PAGE.
Purity >98%, 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 is ≤.2 ng/ml, corresponding to a specific activity of ≥1 x units/mg.
Protein Sequence MAASGITSLP ALPEDGGAAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS SWYVALKRTG QYKLGSKTGP GQKAILFLPM SAKS
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 Recombinant mouse basic FGF 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.


Biological Process Angiogenesis
Biological Process Differentiation
Molecular function Developmental-protein
Molecular function Growth-factor
Molecular function Heparin-binding

Methods

Neurosphere culture

  • Neurospheres were generated from cells isolated from the subventricular zone of 6–8 week old C57BL/6 mice and cultured in proliferation media consisting of DMEM/F12 (GibcoBRL/Invitrogen) containing 0.6% glucose, 3 mM NaHCO3, 5 mM HEPES, 2 mM L-glutamine, 0.1 mg/ml apo-transferrin, 25 µg/ml insulin, 60 µM putrescene, 30 mM sodium selenite, 20 nM progesterone and 1% BSA (all from), supplemented with EGF (20 ng/ml) and FGF2 (10 ng/ml), as previously described

Cell culture

  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with medium'>serum-free medium'>maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40…
  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with medium'>serum-free medium'>maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40 ng/ml bFGF , and 10 ng/ml leukemia inhibitory factor (LIF).
  • All colony-derived cells were re-seeded on new dishes after they reached 90–100% confluence and were maintained with expansion medium.

Read more

Cell culture

  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with medium'>serum-free medium'>maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40…
  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with medium'>serum-free medium'>maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40 ng/ml bFGF , and 10 ng/ml leukemia inhibitory factor (LIF).
  • All colony-derived cells were re-seeded on new dishes after they reached 90–100% confluence and were maintained with expansion medium.

Read more

Soluble amyloid precursor protein alpha (sAPPα) enhances proliferation in an EGF/bFGF-independent manner.

bFGF
  • (b) A neurosphere formation assay was performed without growth factors (EGF and bFGF), and NPCs were treated with an inactive inhibitor , GM6001 , or GM6001 + recombinant sAPPα (GM+rec sAPPα).

Differentiation of radial glia-like cells

  • The development of oligodendrocytes was reached by a 4+4-day protocol, according to Glaser et al., 2007.
  • Briefly, the cells were cultured in basal RGl-medium supplemented with FGF2 (10ng/ml), PDGF (10ng/ml) and forskolin (10 µM), for 4 days.
  • The medium was then replaced with DMEM/F12 (1/1) containing 3,3,5-triiodothyronine (T3; 30 ng/ml) and ascorbic acid (200 µM) as only supplements.
  • At the end of the 8th day, the presence of oligodendrocytes was checked by immunocytochemical staining.

Flow cytometry

  • Differentiated celltype'>hESC cell derived neural induction cultures were rinsed with PBS, dissociated with a 1∶1 mixture of accutase and accumax, filtered through 100 um mesh to remove cell clumps and an aliquot counted on a hemocytometer.
  • Antibodies (BD) were added to a final cell concentration of 1–5×107cells/ml.
  • Labeled cells were sorted at 2.5–5.0×106 cells/ml in neural precursor sort media ( media - DMEM/F12, , , penicillin/streptomycin (all from) and 20 ng/ml bFGF - supplemented with 10% FBS and 0.5 mM EDTA).
  • To estimate the fraction of dead cells, separate aliquots of cells were stained with 750 nM propidium iodide, a membrane impermeable DNA binding dye, and analyzed by flow cytometry.
  • Two to three percent of the cells were propidium iodide positive regardless of the cell line or neural induction method employed.

Primary SV cells

  • Adipose SV cells were isolated from pooled interscapular, inguinal, and epididymal WATs of 5-week-old (P36 or P37) male mice as previously reported

iPSC generation

  • The four mouse reprogramming factors inserted into the pMXs vector backbone were obtained from Addgene (pMXs-cMyc #13375, pMXs-Klf4 #13370, pMXs-Oct4 #13366, pMXs-Sox2 #13367)−1 polybrene (Millipore #Tr-1003-G) twice on consecutive days, then lifted with Accutase (Invitrogen) a day later and replated on irradiated SNL feeders (available from European Collection of Cell Cultures, ECACC).
  • The day after replating, media was switched to hESC medium (KO-DMEM, 20% KSR, 2 mM −1 −1 FGF2 containing 0.5 mM valproate −1 FGF2.

Monolayer neuronal differentiation

  • iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
  • The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
  • kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
  • After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing…
  • iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
  • The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
  • kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
  • After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing the same ligands over 7 days (75% medium'>medium'>KSR and 25% medium'>N2B27 first day, 50% of each third day, and 25% medium'>medium'>KSR with 75% medium'>N2B27 fifth day).
  • Once established in N2B27 conditions, Wnt1, Noggin, SB431542 and −1 BDNF , 0.2 mM ascorbic acid and 100 ng ml−1 FGF8 were added.
  • Three days later, cells were dissociated with Hank's buffered saline solution for 1 h at room temperature, and lifted mechanically, then replated en bloc on to poly-l-ornithine/laminin-coated plasticware.
  • Neuronal maturation ensued with BDNF and ascorbic acid as before, supplemented with 10 ng ml−1 GDNF , 1 ng ml−1 TGFβ3 and 0.5 mM dibutyryl-cAMP for the next 7 days.
  • After a total of 23–31 days, the resulting neuronal cultures were analysed by immunocytochemistry, qPCR and western blot.

Read more