Brain derived neurotrophic factor (bdnf)

Manufactured by ProSpec

Sourced in Germany, Estonia, United States, Israel

BDNF is a laboratory reagent used in research applications. It is a protein that plays a crucial role in the growth, development, and maintenance of neurons in the central and peripheral nervous systems. BDNF is commonly used in neurological and neuroscientific research to study its effects on neuronal function and plasticity.

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Lab products found in correlation

Corticosterone 4 pregnen 11b diol 3 20 dione 21 hemisuccinate, by Merck Group (2 mentions) Hydroxypropyl β cyclodextrin, by Merck Group (2 mentions) Serotonin 5 ht, by Merck Group (2 mentions) Ru486, by Merck Group (2 mentions) Corticosterone, by Merck Group (2 mentions) Fluoxetine, by Merck Group (2 mentions) Cysteamine, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Sh sy5y neuroblastoma cells, by American Type Culture Collection (2 mentions) Retinoic acid, by Merck Group (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Axiocam erc5, by Zeiss (1 mentions) Axio vert a1 microscope, by Zeiss (1 mentions) Digital sight ds fi1, by Nikon (1 mentions) Tryple select, by Thermo Fisher Scientific (1 mentions) Eclipse te2000 s, by Nikon (1 mentions) Mdl28170, by Bio-Techne (1 mentions) Sh sy5y cells, by Merck Group (1 mentions) Y 27632, by Enzo Life Sciences (1 mentions)

10 protocols using brain derived neurotrophic factor (bdnf)

Differentiation of NPCs into Brain Organoids

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NPCs were differentiated into brain organoids according to published protocols [50 (link), 51 (link)] with minor modifications. NPCs were dissociated with accutase (Sigma-Aldrich), briefly centrifuged, and resuspended in NPC medium. Cells were counted using the TC20 automated cell counter (Bio-Rad) and plated at a density of 9000 cells/well on BIOFLOAT™ 96-well plates (faCellitate, F202003). The plate was centrifuged at 220 × g for 5 min and placed in the incubator at 37 °C/5% CO₂. After 48 h, the medium was switched to patterning medium (N2B27 supplemented with 200 μM AA, 1 ng/mL BDNF (Prospec, CYT-207), 1 ng/mL GDNF (PeproTech, 450-10), and 1 µM SAG). After 2 days, the brain organoid media was refreshed with patterning medium. After 2 days, the medium was replaced with maturation medium (N2B27 supplemented with 200 μM AA, 2 ng/mL BDNF, 2 ng/mL GDNF, 1 ng/mL TGF-β3 (Prospec, CYT-368), 5 ng/mL ActivinA (Stem Cell Technologies, 78001), and 100 μM dbcAMP (Sigma-Aldrich, D0260). ActivinA concentration was lowered to 2 ng/mL for the subsequent media changes. The medium was changed every other day. On day 30, the medium was switched to N2B27 without supplements and refreshed every other day.

Carreras Mascaro A., Grochowska M.M., Boumeester V., Dits N.F., Bilgiҫ E.N., Breedveld G.J., Vergouw L., de Jong F.J., van Royen M.E., Bonifati V, & Mandemakers W. (2024). LRP10 and α-synuclein transmission in Lewy body diseases. Cellular and Molecular Life Sciences: CMLS, 81(1), 75.

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3D Cell Culture and Imaging

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Cells were plated either as mechanically cut small cell aggregates or as enzymatically dissociated single cell suspensions prepared using 1X TrypLE Select (Gibco). For the 2D experiments (controls, cells embedded or on top), the plating density was 60,000 cells/cm 2 or 7-20 small aggregates/cm 2 (3000-7000 cells/aggregate). The cell density for the 3D experiments (cells encapsulated) was ~3.5 × 10 6 cells/ml of gel, or a corresponding amount of small mechanically cut cell aggregates.
The cells were cultured with the gel for 2 weeks. NDM without bFGF was used during the first week of the experiments. After one week of culture, NDM containing 5 ng/ml bFGF and 4 ng/ml brain-derived neurotrophic factor (BDNF, Prospec Bio, Germany) was used. Half of the medium was changed three times per week using caution to avoid disrupting the gels.
Cells were imaged using a Zeiss AxioVert.A1 microscope and AxioCam ERc 5s camera system or with a Nikon Eclipse TE 2000-S and Nikon Digital Sight DS-Fi1 camera system during the culturing period.

Koivisto J.T., Joki T., Parraga J.E., Pääkkönen R., Ylä-Outinen L., Salonen L., Jönkkäri I., Peltola M., Ihalainen T.O., Narkilahti S, & Kellomäki M. (2017). Bioamine-crosslinked gellan gum hydrogel for neural tissue engineering. Biomedical materials (Bristol, England), 12(2).

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Hippocampal Slice Cultures: Pharmacological Modulation

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Coronal 300μm slices at the level of the hippocampus were collected using a vibratome (Leica) from freshly isolated P6 sham and TSHI brains. Three slices were then placed on each PTFE insert (0.4μm pore, 30mm diameter, Millipore) and incubated overnight in basal media with 15% fetal calf serum to equilibrate. Each experiment was performed three times using 3 brains total per condition. On day one in vitro (DIV1, P7 equivalent), media was changed to serum free with addition of the following: BDNF (100 ng/ml, Prospec), K252a (0.2 μM, Tocris), AP5 (100μm, Tocris), MDL-28170 (100 μM, Tocris), NMDA (100 μM, Tocris), EPO (10 U/mL, R&D Tissue Grade), polyclonal EPOR neutralizing antibodies (1:50, SantaCruz), or polyclonal isotype control antibodies (1:50, Santa Cruz). Microdissected CA3 was then collected at either 4h or 48h following the administration of these pharmacological agents and processed for Western blots.

Jantzie L.L., Getsy P.M., Firl D.J., Wilson C.G., Miller R.H, & Robinson S. (2014). Erythropoietin Attenuates Loss of Potassium Chloride Co-Transporters Following Prenatal Brain Injury. Molecular and cellular neurosciences, 0, 152-162.

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Intracerebroventricular Injection for SAH Model

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An intracerebroventricular injection procedure was performed, as reported previously (Chen et al., 2013 (link); Suzuki et al., 2010 (link)). A small burr hole was drilled into the skull according to the following coordinates relative to bregma: 1.5 mm posterior; 1.0 mm lateral. The needle of 10 μl Hamilton syringe (Microliter 701; Hamilton Company, Reno, NV) was stereotactically inserted into the left lateral ventricle through the burr hole, 4.0 mm below the horizontal plane of bregma.
Five microliters of HIOC (Vitas-M Laboratory, Narva, Estonia) or BDNF (ProSpec-Tany Technogene, East Brunswick, NJ) in vehicle were infused at a rate of 0.5μl/min 3 hours after SAH induction, while 500pmol/5μl TrkB or scramble siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA) were infused at the same rate 24 hours before SAH modeling. TrkB siRNA is a pool of three different siRNA duplexes in order to improve the knockdown efficiency. All TrkB siRNA sequences are provided in 5′→3′ orientation: (a) sense, GGAUUCCGGCUUAAAGUUU, antisense, AAACUUUAAGCCGGAAUCC; (b) sense, GGAUUUGUAUUGCCUCAAU, antisense, AUUGAGGCAAUACAAAUCC; (c) sense, CCAUCACAUUUCUCGAAUC, antisense, GAUUCGAGAAAUGUGAUGG. The syringe was left in situ for an additional 10 minutes before slowly removing it. In Experiment 1, the sham group rats were subjected to the same procedure, without inserting the needle.

Tang J., Hu Q., Chen Y., Liu F., Zheng Y., Tang J., Zhang J, & Zhang J.H. (2015). Neuroprotective Role of an N-acetyl Serotonin Derivative via Activation of Tropomyosin-related Kinase Receptor B after Subarachnoid Hemorrhage in a Rat Model. Neurobiology of disease, 78, 126-133.

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SH-SY5Y Cell Differentiation Protocol

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SH-SY5Y neuroblastoma cells, obtained from American Type Culture Collection (Virginia, USA) (ATCC Cat. no. CRL-2266, RRID:CVCL_0019), were grown in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Cat. no. 11965092) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Cat. no. 10270098) and 10% Pen-Strep. Cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere. For differentiation, SH-SY5Y cells were grown in DMEM supplemented with 10% FBS and 10 μM RA (Sigma, Cat. no. R2625) for 5 days. At day 5, cell media was changed to DMEM deprived of glucose, pyruvate, and FBS (Thermo Fisher, Cat. no. 11966025), which was supplemented with either 25 mM glucose, 10 mM glucose or 10 mM pyruvate, and 25 ng/mL of BDNF (Prospec, Cat no. CYT-207). Cells were maintained in culture for three more days before the screening or luteolin treatments.

Naia L., Pinho C.M., Dentoni G., Liu J., Leal N.S., Ferreira D.M., Schreiner B., Filadi R., Fão L., Connolly N.M., Forsell P., Nordvall G., Shimozawa M., Greotti E., Basso E., Theurey P., Gioran A., Joselin A., Arsenian-Henriksson M., Nilsson P., Rego A.C., Ruas J.L., Park D., Bano D., Pizzo P., Prehn J.H, & Ankarcrona M. (2021). Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling. BMC Biology, 19, 57.

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Neuronal Conversion of Glioblastoma Cells

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For neuronal conversion, GBM cells were plated at a density of 3.0 × 10⁴ cells cm^− 2 on microscope glass coverslips coated with matrigel (BD) in 35 mm dishes. For neural induction, the media was changed to defined induction medium including DMEM/F12 (2% FBS) plus 1 μM dexamethasone (Millipore-Sigma), 0.5 mM isobutylmethylxanthine (Millipore-Sigma), 200 μM indomethacin (Millipore-Sigma), 2 μM Y-27632 (Enzo Life Sciences) and 2 μM P529 (Millipore-Sigma). For kinase inhibitor screening experiments, we used 2 μM protein kinase inhibitor from a library (Calbiochem, 355 inhibitors). For neuronal differentiation, we used neuronal mature medium including Neural medium (ScienCell) with 50 μM dbcAMP (Millipore-Sigma), 10 ng/ml NT3 (PROSPEC), 10 ng/ml BDNF (PROSPEC), 0.5 μM Retinoic acid (Millipore-sigma), 2 μM Y-27632 and 2 μM P529. Quantitative of mean % induced neuron (iN) purity is counted by morphology of MAP2-positive staining, and numbers represent the percentage of iN cells at the time point of quantification.

Yuan J., Zhang F., Hallahan D., Zhang Z., He L., Wu L.G., You M, & Yang Q. (2018). Reprogramming glioblastoma multiforme cells into neurons by protein kinase inhibitors. Journal of Experimental & Clinical Cancer Research : CR, 37, 181.

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Corticosterone-Induced Neuronal Responses

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NSC 23766 (NSC, Tocris) and Fluoxetine (Sigma, St Louis, MO, USA) were administrated intraperitoneally (i.p; 100 μL) at a concentration of 2.5 mg per kg body weight per day (mg kg^-1 day^-1) and 20 mg kg^-1 day^-1 respectively. Cysteamine (Sigma) was administrated at a concentration of 150 mg kg^-1 day^-1 through drinking water for 21 days. corticosterone (4-pregnen-11b-diol-3 20-dione 21-hemisuccinate; Sigma) was dissolved in vehicle (0.45% hydroxypropyl-β-cyclodextrin, Sigma). corticosterone (35 μg/mL, equivalent to 5 mg kg^-1 day^-1) was delivered ad libitum in the drinking water. The dose and duration of corticosterone treatment in mice were selected based on earlier studies^{9 (link),25 (link)}, where the above dose and duration of treatment with CORT induced anxiety and depressive-like behaviors in mice. The fluid intake was monitored throughout the treatment period and the corticosterone dose was adjusted accordingly. Primary cortical neurons were treated with serotonin (5-HT; Sigma) at a concentration of 5-100 μM at DIV 5-7. In glucocorticoid receptor inhibitor studies, cells were treated with RU486 (1 μM; Sigma) 30 min before 48-h corticosterone (1 μM; Sigma) treatment. Neurons were treated with BDNF (prospec) at 100ng/ml for indicated time period.

Pandya C.D., Hoda N., Crider A., Peter D., Kutiyanawalla A., Kumar S., Ahmed A.O., Turecki G., Hernandez C.M., Terry AV J.r, & Pillai A. (2016). Transglutaminase 2 overexpression induces depressive-like behavior and impaired TrkB signaling in mice. Molecular psychiatry, 22(5), 745-753.

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Corticosterone-Induced Neuronal Responses

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Neuronal Differentiation of SH-SY5Y Cells

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Commercially available SH-SY5Y neuroblastoma cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (Life Technologies, USA) containing fetal bovine serum (FBS) [10%] (Sigma, AUS), L-Glutamine [2 mM] (Life Technologies, USA), penicillin [100 U/ml] and streptomycin [0.1 mg/ml] (Life Technologies, USA). For neuronal differentiation, SH-SY5Y cells were differentiated in complete media with retinoic acid [10µM] (Sigma, USA) for 7 days, followed by a serum-free media containing brain derived neurotropic factor (BDNF) [50 ng/ml] (Prospec Bio, USA) for 5 days. This double differentiated protocol for SH-SY5Y cells has been shown to increase the expression of neurofilament, MAP2 and β-III tubulin, as markers indicative of a more mature neuronal phenotype (Agholme et al, 2010 (link); Encinas et al, 2000 (link)).

Lee T.T., Chana G., Gorry P.R., Ellett A., Bousman C.A., Churchill M.J., Gray L.R, & Everall I.P. (2015). Inhibition of Catechol-O-methyl Transferase (COMT) by Tolcapone Restores Reductions in Microtubule-associated Protein 2 (MAP2) and Synaptophysin (SYP) Following Exposure of Neuronal Cells to Neurotropic HIV. Journal of neurovirology, 21(5), 535-543.

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Murine ES-Derived Neural Stem Cell Culture

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Murine ES-derived NSCs of E14Tg2a ES-background and other wildtype and p53-/- NSCs [6] were grown in Euromed-N cell culture medium (Euroclone), supplemented with L-Glutamine and Penicillin/Streptomycin, 1× N2 supplement (Invitrogen) and 20 ng/ml each murine EGF and FGF2 (ProSpec, Israel). For medium modifications, caspase inhibitor Q-VD-OPH (SM Biochemicals), BMP2 (ProSpec, Israel) or fetal calf serum (FCS) were added at 10 µM or 20 ng/ml or 10%, respectively. For neuronal differentiation, cells seeded on 3 µg/ml Laminin were switched to modified medium as in [8] (link): Euromed-N (Euroclone)/Neurobasal (Invitrogen) 1∶3, 0.5× N2 and 1.5× B27 supplement (Invitrogen), 10 ng FGF2 and 20 ng BDNF (ProSpec, Israel). For BCL2 overexpression, a retroviral pBABE-puro vector carrying human BCL2 cDNA (Addgene, plasmid 21144) or empty pBABE-puro vector were transfected into ecotropic 293T Phoenix cells using calcium-phosphate method. NSC at ∼30% confluence were then infected with viral supernatants, supplemented with 8 µg/ml polybrene (Sigma Aldrich) and selected 24 h later with 0.5 µg/ml puromycine (Sigma Aldrich) until no cell death was visually detectable and irradiated.

, & Schneider L. (2014). Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro. PLoS ONE, 9(1), e87228.

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