Neurobasal a media

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Neurobasal A media is a serum-free cell culture medium specifically designed for the growth and maintenance of primary neuronal cells. It supports the survival and differentiation of neurons while minimizing the growth of non-neuronal cells.

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Neurobasal (586 citations) Neurobasal media (543 citations) Neurobasal A (310 citations) Neurobasal-A growth media (3 citations)

87 protocols using neurobasal a media

Expansion and Differentiation of GBM Cells

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All GBM specimens were collected with written informed consent under a protocol approved by the Institutional Review Board of the Samsung Medical Center (2010-04-004, Seoul, Korea). Following signed informed consent, tumor samples were obtained from GBM patients and dissociated into single cells. For in vitro expansion, patient-derived cells were cultured and passaged in Neurobasal A media (Invitrogen, Camarillo, CA) supplemented with B27 and N2 supplements (0.5X each; Invitrogen, Camarillo, CA) as well as recombinant EGF and bFGF (20 ng/ml each; R&D Systems, Minneapolis, MN).[25 (link)] To mimic differentiation of GBM cells, cells were starved in Neurobasal A media without any supplements for 1 day and then replaced with Neurobasal A media containing 5% (forced condition) or 0.1% (mild condition) FBS (GIBCO, USA), respectively.

Lee Y., Kim K.H., Kim D.G., Cho H.J., Kim Y., Rheey J., Shin K., Seo Y.J., Choi Y.S., Lee J.I., Lee J., Joo K.M, & Nam D.H. (2015). FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2. PLoS ONE, 10(10), e0137703.

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Establishing Primary Hippocampal Neuron Cultures

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Neuronal cultures were obtained from postnatal day 0 (P0) to P2 C57BL/6 mouse hippocampi from both sexes. Hippocampi were removed and digested with papain (2mg/mL, Worthington) in Hibernate without Calcium (BrainBits) and 0.1% DNase at 37°C for 10 minutes. Following titration of tissue by sterile Pasteur pipet, dissociated cells were re-suspended in culture media (Neurobasal A media (ThermoFisherSci) supplemented with GlutaMAX, pen-strep, B27, 1mM HEPES, and 10% horse serum) and plated at 30,000 cells/cm² on poly-d-lysine and laminin coated dishes. Cells were maintained in feeding media (Neurobasal A media (ThermoFisher) supplemented with GlutaMAX, pen-strep, B27, and 1mM HEPES) by half media changes every 3–4 days until DIV14-21. Cultures were incubated at 37°C, 5% CO² and 95% relative humidity.

Kragness S., Clark Z., Mullin A., Guidry J, & Earls L.R. (2022). An Rtn4/Nogo-A-interacting micropeptide modulates synaptic plasticity with age. PLoS ONE, 17(6), e0269404.

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Establishing Murine Hippocampal Neuron Cultures

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Neuronal cultures were obtained from postnatal day 0 (P0) to P2 C57BL/6 mouse hippocampi from both sexes. Hippocampi were removed and digested with papain (2mg/mL, Worthington) in Hibernate without Calcium (BrainBits) and 0.1% DNase at 37°C for 10 minutes. Following titration of tissue by sterile Pasteur pipet, dissociated cells were re-suspended in culture media (Neurobasal A media (ThermoFisherSci) supplemented with GlutaMAX, pen-strep, B27, 1mM HEPES, and 10% horse serum) and plated at 30,000 cells/cm 2 on poly-d-lysine and laminin coated dishes. Cells were maintained in feeding media (Neurobasal A media (ThermoFisher) supplemented with GlutaMAX, pen-strep, B27, and 1mM HEPES) by half media changes every 3-4 days until DIV14-21. Cultures were incubated at 37°C, 5% CO 2 and 95% relative humidity.

Kragness S., Clark Z., Mullin A., Guidry J.J., & Earls L.R. (2022). An Rtn4/Nogo-A-interacting micropeptide modulates synaptic plasticity with age.

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Culturing Medulloblastoma Cell Lines

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Daoy medulloblastoma cell lines were obtained from the American Type Culture Collection. Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% FBS (Gibco) and 1% L‐glutamine (Gibco).
MB002 cells have been described previously as derivative of primary medulloblastoma tumor with large‐cell histology and gene expression markers consistent with Group 3 medulloblastoma (Bandopadhayay et al, 2014). MB002 cells were maintained in culture media made up of 1:1 (v/v) DMEM/F‐12 (Gibco) and Neurobasal‐A media (Gibco, #10888‐022) supplemented with HEPES, sodium pyruvate, non‐essential amino acid, L‐glutamine, B27 (Gibco, #12587‐10), 20 ng/ml EGF (Peprotech; #AF‐100‐15), 20 ng/ml basic fibroblast growth factor (Peprotech; #AF‐100‐18b), heparin (07980; Stem Cell), and 10 ng/ml leukemia inhibitory factor (LIF; LIF1010; Millipore). Medulloblastoma‐initiating cells (MICs) derived from postnatal, day 0, mouse cerebellar neural stem cells—transduced with MYCN^T58A alone or those which were further transduced with SOX9 (MIC‐SOX9) from previously published study (Swartling et al, 2012)—were cultured in Neurobasal‐A media (Gibco, #10888‐022) supplemented with L‐glutamine, B27 (Gibco, #12587‐10), 20 ng/ml EGF (Peprotech; #AF‐100‐15), and 20 ng/ml basic fibroblast growth factor (Peprotech; #AF‐100‐18b).

Suryo Rahmanto A., Savov V., Brunner A., Bolin S., Weishaupt H., Malyukova A., Rosén G., Čančer M., Hutter S., Sundström A., Kawauchi D., Jones D.T., Spruck C., Taylor M.D., Cho Y., Pfister S.M., Kool M., Korshunov A., Swartling F.J, & Sangfelt O. (2016). FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma. The EMBO Journal, 35(20), 2192-2212.

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Primary Mouse Neuronal Culture

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Animals were cared for in accordance with the guidelines set forth by the National Institutes of Health regarding the proper treatment and use of laboratory animals and with the approval of Institutional Animal Care and Use Committee of The Scripps Research Institute. Striata of postnatal day 1 C57BL/6 mice were removed and digested at 37°C for 15 min in a final concentration of 0.25% papain and resuspended in neuronal plating media (Neurobasal-A media; Thermo Fisher Scientific), with 5% FBS, 0.5 mM glutamax, and 1% penicillin-streptomycin. Tissues were dissociated by trituration with a pipette. Further, cells were plated in 35-mm glass-bottom dishes (D11140H; Matsunami) coated with 100 µg/ml poly-D-lysine at the density of 2 × 10⁵ cells per dish. Dishes were maintained in a 37°C, 5% CO₂ incubator. After the cells adhered (1–3 h after plating), plating media were replaced with growth media (Neurobasal-A media, 2% B27, 0.5 mM glutamax, and 1% penicillin-streptomycin).

Sharma M, & Subramaniam S. (2019). Rhes travels from cell to cell and transports Huntington disease protein via TNT-like protrusion. The Journal of Cell Biology, 218(6), 1972-1993.

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Culturing and Maintaining Primary Neuronal Cells

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Sodium Chloride (NaCl), magnesium chloride (MgCl₂), calcium chloride (CaCl₂), glucose, ATP disodium salt, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), naloxone hydrochloride and EGTA (ethylene glycol tetra acetic acid) were purchased from Sigma-Aldrich (St. Louis, MO). Potassium chloride (KCl) was purchased from Fisher Scientific (Waltham, MA) and collagenase type II was purchased from Worthington (Lakewood, NJ). Laminin and poly-D-lysine were purchased from BD bioscience (Franklin lanes, NJ); GDNF (glial cell-derived neurotrophic factor) was purchased from Neuromics (Edina, MN); FBS (fetal bovine serum) was purchased from Gemini Bio products (West Sacramento, CA); B-27, trypsin and neurobasal A media were purchased from Thermofisher (Waltham, MA). Morphine sulfate was obtained from National Institutes of Drug Abuse (Bethesda, MD).

Muchhala K.H., Koseli E., Gade A.R., Woods K., Minai S., Kang M., McQuiston A.R., Dewey W.L, & Akbarali H.I. (2022). Chronic Morphine Induces IL-18 in Ileum Myenteric Plexus Neurons Through mu-Opioid Receptor Activation in Cholinergic and VIPergic Neurons. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 17(1-2), 111-130.

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Rat Hippocampal Neuron Culture

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Hippocampi from P0–1 rat pups were prepared as previously described.⁷⁶ Dissociated neurons were plated at a density of 130 cells per mm^{2 (link)} on 12 mm diameter glass coverslips coated with poly-d-lysine (Neuvitro, Vancouver, WA). Neurons were continuously maintained at 37°C and 5% CO2 and grown in maintenance media consisting of Neurobasal-A media, supplemented with Glutamax, and B27 supplement (all from Thermo Fisher Scientific, Waltham, MA).

Campbell E.P., Abushawish A.A., Valdez L.A., Bell M.K., Haryono M., Rangamani P, & Bloodgood B.L. (2023). Electrical signals in the ER are cell type and stimulus specific with extreme spatial compartmentalization in neurons. Cell reports, 42(1), 111943.

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Neurosphere Formation Assay with Metformin

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Mice were anesthetized with isoflurane and euthanized by cervical dislocation. The spinal cord and the PVZ were carefully dissected under a Stemi 2000 microscope (Zeiss, Germany), minced and incubated in papain (Worthington Biochemicals, NJ, USA) dissolved in sterile Earle’s Balanced Salt Solution (EBSS, Worthington Biochemicals). Cells were triturated and spun and then resuspended in a solution containing DNase Ovomucoid protease inhibitor (Worthington Biochemicals), in EBSS and centrifuged. Cells were resuspended in Neurobasal-A media (NBM, Thermo Fisher Scientific, PA, USA) containing l-glutamine (2 mM, Invitrogen, CA, USA), penicillin/streptavidin (100 U/0.1 mg/mL, Invitrogen), epidermal growth factor (EGF, 20 ng/mL; Peprotech, QC, CA); fibroblast growth factor (FGF, 10 ng/mL; Gibco, NY, USA); and heparin (2 µg/mL), Sigma-Aldrich, MI, USA) and centrifuged. Cells were resuspended in culture media in the absence or presence of metformin (0.1, 1, 10, 50, 100, 250, and 500 ng/mL). Cells were plated at a density of 10 cells/µL in 24-well plates (Thermo Fisher Scientific) in media for 7 days at 37 C and 5% CO₂. The numbers of neurospheres (>80 µm) were counted in 6 wells of a 6 well plate (Sigma-Aldrich) and averaged per animal.

Gilbert E.A., Livingston J., Garcia-Flores E., Kehtari T, & Morshead C.M. (2023). Metformin Improves Functional Outcomes, Activates Neural Precursor Cells, and Modulates Microglia in a Sex-Dependent Manner After Spinal Cord Injury. Stem Cells Translational Medicine, 12(6), 415-428.

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Dissociated Hippocampal Neuronal Culture

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All animal procedures were performed in accordance with the University of California San Diego Institutional Animal Care and Use Committee and complied with all relevant ethical regulations for animal research. Dissociated hippocampal neuronal cultures were derived from wild-type Sprague Dawley rat pups of both sexes at postnatal days P0–P1. The cell cultures were prepared as previously described (Goslin et al., 1991 ), and plated at a density of 130 cells per mm² on laminin-coated 35-mm MatTek dishes (MatTek CatP35G-0-14-C). Neurons were grown in Neurobasal-A media (ThermoFisher Scientific Cat10888022) supplemented with Glutamax (ThermoFisher Scientific Cat35050061), Pen/Strep (ThermoFisher Scientific Cat10378016), and B27 supplement (ThermoFisher Scientific Cat17504044).

Puppo F., Sadegh S., Trujillo C.A., Thunemann M., Campbell E.P., Vandenberghe M., Shan X., Akkouh I.A., Miller E.W., Bloodgood B.L., Silva G.A., Dale A.M., Einevoll G.T., Djurovic S., Andreassen O.A., Muotri A.R, & Devor A. (2021). All-Optical Electrophysiology in hiPSC-Derived Neurons With Synthetic Voltage Sensors. Frontiers in Cellular Neuroscience, 15, 671549.

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Isolation and Culture of Cortical Astrocytes and Neurons

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Purified cortical astrocyte cultures were prepared from post-natal days 3-4 rat pups. In brief, cortical cells were disassociated and then suspended in Neurobasal A media (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum, and 1% Glutamax (Thermo Fisher Scientific, Waltham, MA). Cells were initially grown in 25cm² or 75cm² cell culture flasks. Once confluent, cells were then subjected to prolonged, orbital shaking (250 rpm for 18 hours at 37°C) which has been used to yield purified astrocyte cultures (McCarthy and de Vellis, 1980 (link)). Purified astrocytes were then plated on 24-well plates, and refreshed with 70% new culture media every 2-3 days.
Purified cortical neuronal cultures were prepared from embryonic rat cortical tissue (gestational day 15 to 16) as previously described (Lobner, 2000 (link)). In brief, dissociated cells were suspended in Eagles’ Minimal Essential Medium (MEM, Earle’s salts, glutamine-free) supplemented with glutamine (2mM), glucose (21mM), horse serum (5%), and fetal bovine serum (5%). Cells were seeded on 24-well plates. Forty eight hours later, cytosine arabinoside (at a final concentration of 10 μM) was added to the culture media to inhibit glial reproduction (Dugan et al., 1995 (link)). Neurons were then grown for an additional 11-13 days.

Kong L., Albano R., Madayag A., Raddatz N., Mantsch J.R., Choi S., Lobner D, & Baker D.A. (2016). Pituitary Adenylate Cyclase-Activating Polypeptide Orchestrates Neuronal Regulation of the Astrocytic Glutamate Releasing Mechanism System xc−. Journal of neurochemistry, 137(3), 384-393.

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