Human Brain-Derived Neurotrophic Factor Recombinant

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Human Brain-Derived Neurotrophic Factor Recombinant

$70.00$4,700.00


accession P23560


Source Optimized DNA sequence encoding Human Brain Derived Neurotrophic Factor mature chain was expressed in Escherichia Coli.
Molecular weight Human native BDNF is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately 14 kDa. Recombinant human BDNF is a disulfide-linked homodimeric protein consisting of two 119 amino acid residue subunits. migrates as an approximately 27 kDa protein under non-reducing conditions and as a 14 kDa protein under reducing conditions in SDS-PAGE.
Purity >97%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) was determined by the dose-dependent proliferation of rat C6 cells, and was found to be in the range of 0.5 ug/ml.

Protein Sequence MTILFLTMVI SYFGCMKAAP MKEANIRGQG GLAYPGVRTH GTLESVNGPK AGSRGLTSLA DTFEHVIEEL LDEDQKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSMRVRRHS DPARRGELSV CDSISEWVTA ADKKTAVDMS GGTVTVLEKV PVSKGQLKQY FYETKCNPMG YTKEGCRGID KRHWNSQCRT TQSYVRALTM DSKKRIGWRF IRIDTSCVCT LTIKRGR
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 Brain Derived Neurotrophic Factorwas lyophilized from a.2 μm filtered citric acid, pH.7.
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 P20783 NTF3_HUMAN
Interactor P35739 NTRK1_RAT
Interactor Q16620 NTRK2_HUMAN
Interactor Q99523
Molecular function Growth-factor

Methods

Neural induction

  • After 6-8 (early) and 20-30 (late) passages, iPSC and late-passage (30-40) ESCs were subjected to neural differentiation according to a previously established procedure for ESCs (Figure 4 (iPSC) or 5 × 104 (ESC) cells per ml to allow embryoid body (EB) formation.
  • Differentiation medium was changed at day 3.
  • On day 5, EBs were plated en bloc on tissue culture plates or chamber slides double-coated with poly-D-lysine (200 μg/ml) and mouse laminin (10 μg/ml) at a density of 1-2 × 102 EBs per cm2 in fresh medium.
  • Before plating, EB were imaged to assess size and shape.
  • At least 50 EBs were analyzed using MetaMorph software to determine the average EB diameter for each biological replicate.
  • Twenty-four-thirty-six hours post plating, the medium was changed to neural induction medium containing GMEM, 1% N2, 2 mM glutamine, 1 mM sodium pyruvate,…
  • After 6-8 (early) and 20-30 (late) passages, iPSC and late-passage (30-40) ESCs were subjected to neural differentiation according to a previously established procedure for ESCs (Figure 4 (iPSC) or 5 × 104 (ESC) cells per ml to allow embryoid body (EB) formation.
  • Differentiation medium was changed at day 3.
  • On day 5, EBs were plated en bloc on tissue culture plates or chamber slides double-coated with poly-D-lysine (200 μg/ml) and mouse laminin (10 μg/ml) at a density of 1-2 × 102 EBs per cm2 in fresh medium.
  • Before plating, EB were imaged to assess size and shape.
  • At least 50 EBs were analyzed using MetaMorph software to determine the average EB diameter for each biological replicate.
  • Twenty-four-thirty-six hours post plating, the medium was changed to neural induction medium containing GMEM, 1% N2, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 0.01% penicillin streptomycin and 10 ng/ml brain-derived neurotrophic factor (BDNF) .
  • Neural induction cultures were maintained for 3, 7 or 15 days before cells were harvested for RNA extraction, electrophysiological recordings, flow cytometry analysis, or fixation with 4% paraformaldehyde for immunocytochemistry.

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Acute BDNF treatment induces maturation of the dendritic spine population.

BDNF
  • 11 DIV hippocampal neurons were transfected with a vector expressing Lifeact-ruby, and 24 hrs later, the neurons were treated with vehicle or 100 ng/ml BDNF followed by time-lapse imaging every 5 minutes for 1 hr.

Neural precursor culture and viral transduction

  • To differentiate NPs to neurons, NPs were plated on polyornithine and laminin coated plates in NP media and grown until they reached 70% confluence.
  • FGF was removed and NP media supplemented with 20 ng/ml BDNF , 20 ng/ml GDNF and 0.5 mM dibutyryl cAMP (N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt).
  • Medium was exchanged every 2–3 days.

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.

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Preparation of Primary Hippocampal Cultures

  • Primary hippocampal cultures were prepared from wild-type neonatal (E19) rat embryos (timed pregnant Sprague Dawley rats were obtained from Charles River Laboratories, Wilmington, MA) as described previously

Developmental regulation of BDNF-induced TrkB receptor phosphorylation.

BDNF
  • (b) Representative blots of experiments showing that BDNF (50 ng/ml, 15 min, 37°C) readily induces TrkB phosphorylation at sites Y515 and Y705/6 in P8 hippocampal microslices whereas neither site is effectively phosphorylated by BDNF in P24 hippocampal microslices.

Immunoisolation and Culture of RGC from Postnatal Mouse Retinae

  • Seven days old mice were killed according to institutional guidelines.
  • To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
  • The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
  • Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
  • Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
  • Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
  • Positive cell isolation was then achieved by incubating the filtrated…
  • Seven days old mice were killed according to institutional guidelines.
  • To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
  • The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
  • Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
  • Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
  • Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
  • Positive cell isolation was then achieved by incubating the filtrated supernatant for 45 min on a 100-mm diameter petri dish sequentially pre-coated with goat anti-mouse-IgG and anti-Thy1.2 (mouse IgM, clone F7D5, , ).
  • Non-adherent cells were thoroughly washed off, and the bound cells were released by trypsination (12,000 U/ml in Hank’s buffered salt solution [HBSS] for 10 min in 5% CO2 at 37°C) and resuspended in culture medium.
  • The average yield, in thousands, of RGC per animal was 27.8 (±3.3), n = 11.
  • RGC were plated at 600 cells mm−2 on glass coverslips (10 mm in diameter) centered on 12-well tissue culture plates , which were pre-coated with 5 μg/ml poly-d-lysine (MW–40 kDa).
  • Neurons were cultured for a week at 37°C, 5% CO2, 95% humidity in medium'>Neurobasal medium supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and pyruvate (1 mM), glutamine (2 mM), N-acetyl-l-cysteine (60 μg/ml), putrescine (16 μg/ml), selenite (40 ng/ml), bovine serum albumin (100 μg/ml; fraction V, crystalline grade), triiodothyronine (40 ng/ml), holotransferrin (100 μg/ml), dibutyryl cyclic AMP (250 μM), insulin (5 μg/ml), progesterone (62 ng/ml), B27 (1:50), d-mannose (50 μM), brain-derived neurotrophic factor (BDNF; 25 ng/ml, , ), ciliary neurotrophic factor (CNTF; 10 ng/ml) and forskolin (10 μM).
  • Cell viability was checked by examining the cell cultures under a phase contrast microscope (AxioVert25, Carl Zeiss, Jena, Germany) and determining cell adherence to the cover slip and the fraction of cells developing neurites.
  • We waited a week in order to let RGC develop a massive neuritic arborisation.
  • Since we were interested in the effect of PEDF on neuronal survival rather than on neurite growth, only preparations exhibiting an important development of neurites were selected for further tests.

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Immunoisolation and Culture of RGC from Postnatal Mouse Retinae

  • Seven days old mice were killed according to institutional guidelines.
  • To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
  • The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
  • Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
  • Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
  • Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
  • Positive cell isolation was then achieved by incubating the filtrated…
  • Seven days old mice were killed according to institutional guidelines.
  • To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
  • The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
  • Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
  • Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
  • Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
  • Positive cell isolation was then achieved by incubating the filtrated supernatant for 45 min on a 100-mm diameter petri dish sequentially pre-coated with goat anti-mouse-IgG and anti-Thy1.2 (mouse IgM, clone F7D5, , ).
  • Non-adherent cells were thoroughly washed off, and the bound cells were released by trypsination (12,000 U/ml in Hank’s buffered salt solution [HBSS] for 10 min in 5% CO2 at 37°C) and resuspended in culture medium.
  • The average yield, in thousands, of RGC per animal was 27.8 (±3.3), n = 11.
  • RGC were plated at 600 cells mm−2 on glass coverslips (10 mm in diameter) centered on 12-well tissue culture plates , which were pre-coated with 5 μg/ml poly-d-lysine (MW–40 kDa).
  • Neurons were cultured for a week at 37°C, 5% CO2, 95% humidity in medium'>Neurobasal medium supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and pyruvate (1 mM), glutamine (2 mM), N-acetyl-l-cysteine (60 μg/ml), putrescine (16 μg/ml), selenite (40 ng/ml), bovine serum albumin (100 μg/ml; fraction V, crystalline grade), triiodothyronine (40 ng/ml), holotransferrin (100 μg/ml), dibutyryl cyclic AMP (250 μM), insulin (5 μg/ml), progesterone (62 ng/ml), B27 (1:50), d-mannose (50 μM), brain-derived neurotrophic factor (BDNF; 25 ng/ml, , ), ciliary neurotrophic factor (CNTF; 10 ng/ml) and forskolin (10 μM).
  • Cell viability was checked by examining the cell cultures under a phase contrast microscope (AxioVert25, Carl Zeiss, Jena, Germany) and determining cell adherence to the cover slip and the fraction of cells developing neurites.
  • We waited a week in order to let RGC develop a massive neuritic arborisation.
  • Since we were interested in the effect of PEDF on neuronal survival rather than on neurite growth, only preparations exhibiting an important development of neurites were selected for further tests.

Read more

Culture and Differentiation of Human PSCs

  • Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
  • The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
  • Culture medium was changed every day and cells were passaged every 2–3 days.
  • If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
  • For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
  • Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
  • Media changes were…
  • Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
  • The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
  • Culture medium was changed every day and cells were passaged every 2–3 days.
  • If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
  • For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
  • Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
  • Media changes were performed every two days.
  • For differentiation to midbrain dopaminergic (A) neurons and spinal motor neurons, hNPCPSA-NCAM+ were treated with 200 ng/ml Shh and 100 ng/ml FGF8 or 100 ng/ml Shh and 0.5 µM retinoic acid (A) for 7 or 8 days, respectively.
  • The cells were re-plated on new Matrigel-coated dishes at a low density and cultured in differentiation media supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF) , and 10 ng/ml glial cell-derived neurotrophic factor (GDNF) for 3 weeks.

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Culture and Differentiation of Human PSCs

  • Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
  • The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
  • Culture medium was changed every day and cells were passaged every 2–3 days.
  • If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
  • For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
  • Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
  • Media changes were…
  • Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
  • The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
  • Culture medium was changed every day and cells were passaged every 2–3 days.
  • If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
  • For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
  • Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
  • Media changes were performed every two days.
  • For differentiation to midbrain dopaminergic (A) neurons and spinal motor neurons, hNPCPSA-NCAM+ were treated with 200 ng/ml Shh and 100 ng/ml FGF8 or 100 ng/ml Shh and 0.5 µM retinoic acid (A) for 7 or 8 days, respectively.
  • The cells were re-plated on new Matrigel-coated dishes at a low density and cultured in differentiation media supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF) , and 10 ng/ml glial cell-derived neurotrophic factor (GDNF) for 3 weeks.

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