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Brain derived neurotrophic factor (bdnf)

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BDNF is a laboratory product offered by Miltenyi Biotec. It is a recombinant protein that functions as a growth factor. The core function of BDNF is to promote the survival and growth of certain types of neurons.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Retinoic acid, by Merck Group (2 mentions) Forskolin, by Bio-Techne (1 mentions) Pd0332991, by Bio-Techne (1 mentions) Lm22a4, by Bio-Techne (1 mentions) Chir99021, by Bio-Techne (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Relesr, by STEMCELL (1 mentions) Macs ips brew media, by Miltenyi Biotec (1 mentions) Purmorphamine, by Miltenyi Biotec (1 mentions) Ldn 193189, by Miltenyi Biotec (1 mentions) Chir99021, by Miltenyi Biotec (1 mentions) Retinoic acid, by Cayman Chemical (1 mentions) Sb431542, by Cayman Chemical (1 mentions) Ldn 193189, by Cayman Chemical (1 mentions) Fibroblast growth factor 2 (fgf2), by Miltenyi Biotec (1 mentions) Purmorphamine, by Cayman Chemical (1 mentions) Sb431542, by Abcam (1 mentions) N2 supplement, by Thermo Fisher Scientific (1 mentions) Matrigel, by Thermo Fisher Scientific (1 mentions)

9 protocols using brain derived neurotrophic factor (bdnf)

1

Neuronal Differentiation and Maturation Protocol

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Stage 3 astrocytes were plated and differentiated for 1 week in Stage 4 medium before seeding the NPCs on top. Dissociated NPCs were plated at a density of 25,000 per coverslip on top of confluent Stage 4 astrocytes. From this point, only neuronal differentiation/maturation media were used, with no further supplements for the astrocytes.
The neuronal/astrocyte co-cultures were maintained using theSynaptoJuice Protocol (Kemp et al., 2016 (link)). During the first week, thecells were differentiated in SynaptoJuice-A medium [Advanced DMEM/F12 1:1 Neurobasal A, 1% Glutamax, 1% antibiotic/antimyotic, 2% B27 without vitamin A, 2 μM PD0332991 (Tocris), 10 μM DAPT, 10 ng/mL BDNF (Miltenyi), 500 nM LM22A4 (Tocris), 10 μM forskolin (Tocris), 3 μM CHIR99021 (Tocris), 300 μM GABA (Tocris), 1.8 mM CaCl2 (Sigma), 200 μM ascorbic acid (Sigma)]. SynpatoJuice-A was changed on day 2 and day 5 after plating. On day 7 post-plating, SynaptoJuice-A was replaced with SynaptoJuice-B differentiation/maturation medium (Advanced DMEM/F12 1:1, Neurobasal A, 2% Glutamax, 1% antibiotic/antimyotic, 2% B27, 2 μM PD0332991, 10 ng/mL BDNF, 500 nM LM22A4, 3 μM CHIR99021, 1.8 mM CaCl2, 200 μM ascorbic acid). SynaptoJuice-B was changed every 3 days for up to day 24 days post-plating, at which time the cultures were fixed for immunocytochemistry.
Garcia V.J., Rushton D.J., Tom C.M., Allen N.D., Kemp P.J., Svendsen C.N, & Mattis V.B. (2019). Huntington’s Disease Patient-Derived Astrocytes Display Electrophysiological Impairments and Reduced Neuronal Support. Frontiers in Neuroscience, 13, 669.
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2

Differentiation of iPSCs to Spinal Motor Neurons

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Wild-type BJ fibroblast-derived iPSCs (BJ-iPS) were cultured feeder-free on matrigel-coated plates in MACS iPS-Brew media (Miltenyi Biotec). Routine passaging using ReLeSR (Stem Cell Technologies) was performed once every 6-7 days. Pluripotent stem cells were differentiated towards the spinal motor neuron fate following established protocols described previously. Briefly, we first neutralized the BJ-iPS by activating Wnt pathways with CHIR99021 treatment (4.25 μM, Miltenyi Biotec) while blocking Bone Morphogenic Protein (BMP) signaling by LDN-193189 treatment (0.5 μM, Miltenyi Biotec) at the same time. At day 3, variable concentrations of retinoic acid and GDF11 were added to initiate the rostral-caudal patterning, in the presence of fixed concentration of Purmorphamine (1 μM, Miltenyi Biotec), a Sonic Hedgehog pathway agonist, as a ventralizing signal. Neurotrophic factors, BDNF (20 ng/ml, Miltenyi Biotec) and GDNF (20 ng/ml, Miltenyi Biotec), were added to the neuronal cultures at day 17 to promote neuronal maturation into motor neurons.
Lim G.S., Hor J.H., Ho N.R., Wong C.Y., Ng S.Y., Soh B.S, & Shao H. (2019). Microhexagon gradient array directs spatial diversification of spinal motor neurons. Theranostics, 9(2), 311-323.
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3

Efficient Differentiation of iPSCs into iHTNs

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For differentiation into iHTNs, iPSCs were accutase-treated and plated as single cells in 6-well Matrigel-coated plates at a density of approx. Overall, 1x106 cells per well in E8 medium with ROCK-inhibitor Y27632 (10 μM; Stemgent). The next day iHTN differentiation was initiated by neuroectoderm differentiation by dual SMAD inhibition using LDN193189 (1 μM, Cayman) and SB431542 (10 μM, Cayman) and this treatment is carried on for 48 h. This was followed by Sonic hedgehog activation by Smoothened agonist SAG (1 μM, Tocris) and purmorphamine (PMN, 1 μM, Tocris) and Wnt signaling inhibition using IWR-endo (10 μM, Cayman) from day 3 to 8 to direct the cells towards ventral diencephalon with regular media change every 2 days. From day 9 to 13, the cells are slowly made to exit cell cycle using DAPT (10 μM, Cayman) in the presence of ventralizing agent retinoic acid (0.1 μM, Cayman). On day 14, the cells were treated with Accutase and re-plated onto laminin-coated plates in the presence of maturation medium containing brain-derived neurotrophic factor BDNF (10 ng/mL, Miltenyi) and maintained until day 40.
Rajamani U., Gross A.R., Ocampo C., Andres A.M., Gottlieb R.A, & Sareen D. (2017). RETRACTED ARTICLE:Endocrine disruptors induce perturbations in endoplasmic reticulum and mitochondria of human pluripotent stem cell derivatives. Nature Communications, 8, 219.
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4

Oligodendrocyte, Neuron, and Astrocyte Differentiation

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For oligodendrocytic differentiation, cells were plated onto poly‐ornithine‐/laminin‐coated plates or slides in expansion medium for 24–48 h. Medium was changed to DMEM/F12 supplemented with N2, forskolin (10 nM), FGF2 (10 ng/ml), and PDGF (10 ng/ml) for 5 days. From Day 5, medium was switched to DMEM/F12 with N2, thyroid hormone T3 (30 ng/ml), ascorbic acid (200 μM) all supplements from Stem Cell and PDGF (10 ng/ml) (Miltenyi biotech). On Day 15, PDGF was withdrawn from culture to allow maturation and O4 positive could be detected after 5 weeks of differentiation. For neuronal differentiation, cells were plated on poly‐ornithine‐ and laminin‐coated plates or slides in expansion medium without EGF. After 10 days, medium was switched to neurobasal, with N2, B27, and FGF2; 4 days later, FGF2 was withdrawn and 4 days after that, medium was switched to neurobasal supplemented with B27 + A, CNTF, and BDNF (Miltenyi biotech). For astrocytic differentiation, cells were treated with 5% serum. All medium supplements were from Stem Cell.
Assad Kahn S., Costa S.L., Gholamin S., Nitta R.T., Dubois L.G., Fève M., Zeniou M., Coelho P.L., El‐Habr E., Cadusseau J., Varlet P., Mitra S.S., Devaux B., Kilhoffer M., Cheshier S.H., Moura‐Neto V., Haiech J., Junier M, & Chneiweiss H. (2016). The anti‐hypertensive drug prazosin inhibits glioblastoma growth via the PKCδ‐dependent inhibition of the AKT pathway. EMBO Molecular Medicine, 8(5), 511-526.
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5

BDNF Enhances MC3T3-E1 Cell Proliferation

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To analyze the effects of BDNF on cell proliferation, MC3T3-E1 cells were plated in 6-well plates at density of 5 x 103/well and were incubated in the presence or absence of BDNF (80 ng/ml, Miltenyi Biotec Inc., CA, USA). Firstly, we tried in vitro experiments using 50, 80 and 100 ng/ml of BDNF, and we determined the most effective concentration of BDNF as 80 ng/ml (data not shown). At 1, 3, 5, and 7 days after adding BDNF, the cells were collected by 0.25% trypsin, and the total number of the cells was counted using a hemocytometer. We repeated the same assays more than 3 times for each condition.
Ida-Yonemochi H., Yamada Y., Yoshikawa H, & Seo K. (2017). Locally Produced BDNF Promotes Sclerotic Change in Alveolar Bone after Nerve Injury. PLoS ONE, 12(1), e0169201.
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6

Differentiation of Human Neural Precursors

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Cultures of human neural precursor cells (NPCs) and motor neurons differentiated from H9 hES cell line were prepared as described previously [30 (link)]. Briefly, hES cells were maintained in mTESR2 media (Stemcell Technologies) on Matrigel® (Corning) coated dishes. Confluent hES H9 cultures were switched to differentiation medium composed of ADF supplemented with SB431542 (10 μM, Abcam). Purmorphamine (1 μM, Cayman Chemicals) and retinoic acid (0.1 μM, Sigma) were added on Day 4. On Day 8, cells were split in 1:2 ratio and on Day 16, NPCs were dissociated using Accutase®, plated onto Matrigel® coated dishes and cultured in ADF with GlutaMAX, penicillin-streptomycin, B27 (12587–010) and N2 supplements (all Life Technologies) and BDNF (Miltenyi, 10 ng/ml). On Day 23, Accutase® was used to re-plate neurons on dishes/coverslips at desired density. Neurons were cultured in 50:50 mixture of ADF/Neurobasal A with the above supplements until Day 40.
Shelkovnikova T.A., Kukharsky M.S., An H., Dimasi P., Alexeeva S., Shabir O., Heath P.R, & Buchman V.L. (2018). Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis. Molecular Neurodegeneration, 13, 30.
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7

Neurogenic and Multilineage Differentiation Potential of Stem Cells

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The neurogenic differentiation potential of drNPC was determined by culture in the neuron-induction medium comprising Neurobasal (Gibco, United States), L-glutamine (1X, Gibco), antibiotics (1X, Gibco) and the growth factors BDNF 20 ng/ml and GDNF 20 ng/ml (both from Miltenyi Biotec). The osteogenic, adipogenic and chondrogenic differentiation potential of MSC was determined by culture in an osteogenic medium [DMEM supplemented with 10–8 M dexamethasone (Sigma, D4902), 10 mM b-glycerophosphate (Sigma, G9422), and 50 μg/ml ascorbic acid], adipogenic medium [DMEM supplemented with 10 mM 3 isobutyl-1-methylxanthine (Sigma, 17018), 0.1 mM indomethacin (Sigma, 17378), 10 μg/ml insulin (Sigma, I6634), 10–6 dexamethasone] or chondrogenic medium (Stempro, Invitrogen), respectively, with subsequent verification by staining with Alizarin Red (Sigma), Oil Red O (Sigma) or Alcian Blue (Sigma), respectively. A Nikon Eclipse Ci microscope (Nikon Instruments Inc, Japan) was used for image capture.
Namestnikova D.D., Gubskiy I.L., Revkova V.A., Sukhinich K.K., Melnikov P.A., Gabashvili A.N., Cherkashova E.A., Vishnevskiy D.A., Kurilo V.V., Burunova V.V., Semkina A.S., Abakumov M.A., Gubsky L.V., Chekhonin V.P., Ahlfors J.E., Baklaushev V.P, & Yarygin K.N. (2021). Intra-Arterial Stem Cell Transplantation in Experimental Stroke in Rats: Real-Time MR Visualization of Transplanted Cells Starting With Their First Pass Through the Brain With Regard to the Therapeutic Action. Frontiers in Neuroscience, 15, 641970.
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8

Differentiation of Human Neural Progenitor Cells

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The generation and culturing of human NPCs has been previously described (37 (link)). NPCs were grown in N2B27 medium (DMEM-F12/Neurobasal at 50:50 supplemented with 1% penicillin/streptomycin/glutamine, 1% B27 supplement without vitamin A, and 0.5% N2 supplement) containing 3 μM CHIR99021 (Cayman), 150 μM L-ascorbic acid (Sigma-Aldrich), and 0.5 μM Smoothened Agonist (SAG) (Cayman). For differentiation, the medium was replaced with N2B27 containing 1 ng/ml BDNF (Miltenyi Biotec), 0.2 mM L-ascorbic acid, 1 μM retinoic acid (Sigma-Aldrich), 1 ng/ml glial cell line-derived neurotrophic factor (GDNF) (Miltenyi Biotec), and 0.5 μM SAG. On day 8, the medium was changed for inducing neural maturation to N2B27 containing 5 ng/ml activin A (Miltenyi Biotec), 0.1 mM dbcAMP (Sigma-Aldrich), 2 ng/ml BDNF, 0.2 mM L-ascorbic acid, 1 ng/ml TGFβ-3 (Peprotech), and 2 ng/ml GDNF. On day 10, the cells were seeded on a four-well plate for immunofluorescence and 2.5 μM N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) (Sigma-Aldrich) and activin were added to the maturation medium. After 2 d, DAPT and acitivin were removed and the cells were further grown in the maturation medium for neural maturation until day 30.
Martinez-Macias M.I., Moore D.A., Green R.L., Gomez-Herreros F., Naumann M., Hermann A., Van Damme P., Hafezparast M, & Caldecott K.W. (2019). FUS (fused in sarcoma) is a component of the cellular response to topoisomerase I–induced DNA breakage and transcriptional stress. Life Science Alliance, 2(2), e201800222.
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9

Establishment of Myoblast-Motor Neuron Coculture

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The 96 micropatterned‐well plates were designed and provided by CYTOO SA [23 (link)] (Minatec BHT, Grenoble, France). At D0, myoblasts were seeded at 20,000 cells per well in DMEM‐F12 glutamax (ThermoFisher®) supplemented by 20% foetal bovine serum (Sigma‐Aldrich®) and 0.1% penicillin/streptomycin (ThermoFisher®) (supporting information Material and Method Table S1). At D2, after two washes with basal medium, the growth medium was replaced by the differentiation medium composed of DMEM‐F12 glutamax and 2% horse serum (ThermoFisher®, 26050088). At D4, hiPSC‐derived MNs were seeded at 40,000 cells per well in DMEMF12‐glutamax, neurobasal medium (Vol:Vol) supplemented with BDNF (Miltenyi Biotec®), GDNF (Miltenyi Biotec®), DAPT (Sigma‐Aldrich®) and Y‐27632 (Stem Cell®) (supporting information Material and Method Table S1). Cocultures were maintained for at least 7 days.
Tahraoui‐Bories J., Mérien A., González‐Barriga A., Lainé J., Leteur C., Polvèche H., Carteron A., De Lamotte J.D., Nicoleau C., Polentes J., Jarrige M., Gomes‐Pereira M., Ventre E., Poydenot P., Furling D., Schaeffer L., Legay C, & Martinat C. (2023). MBNL‐dependent impaired development within the neuromuscular system in myotonic dystrophy type 1. Neuropathology and Applied Neurobiology, 49(1), e12876.
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