Neuronal medium

Manufactured by ScienCell

Sourced in United States

Neuronal Medium is a specialized cell culture medium designed to support the growth and maintenance of neuronal cells. It provides the essential nutrients and growth factors required for the optimal proliferation and differentiation of neuronal cells in vitro.

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15 protocols using neuronal medium

Reprogramming Fibroblasts into Neurons

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Fibroblasts were transduced with lentiviruses containing transgenes for miR-9/9*-124, NEUROD2, ASCL1 and MYT1L as previously described.^{9 (link)} Fibroblasts were plated at a density of 10–15 k cm⁻² in fibroblast medium (Dulbecco's Modified Eagle Medium; Invitrogen) containing 10% fetal bovine serum (FBS; Gemini Bio-Products), nonessential amino acids, glutamate and penicillin/streptomycin (Invitrogen) on plates coated with 0.1% gelatin (Millipore, Billerica, MA, USA). Fibroblast medium contained DMEM (Invitrogen) with 10% FBS (Gemini Bio-Products), 1% Pen-Strep (Invitrogen, Grand Island, NY, USA), 1% non-essential amino acids (Invitrogen) and 1% 200 mM L-glutamine (Invitrogen). Infection with the four lentiviruses occurred 24 h after plating using 8 μg ml⁻¹ polybrene (Sigma) with plates centrifuged at 1000 g for 20 min to enhance the lentivirus infection efficiency. The next day, medium was changed to fibroblast medium containing 1 mM valproic acid (Sigma), and 3 days later, the medium was changed to Neuronal Medium (ScienCell, Carlsbad, CA, USA) containing 1 mM valproic acid along with selection antibiotics Geneticin, Blasticidin and Puromycin (all Invitrogen). Selection antibiotics were removed after 6–7 days, and the cells were maintained in Neuronal Medium (ScienCell) with 1 mM valproic acid for 1–3 days until passaging with Accutase (Sigma) onto a 384-well BIND biosensor.

Wang J.L., Shamah S.M., Sun A.X., Waldman I.D., Haggarty S.J, & Perlis R.H. (2014). Label-free, live optical imaging of reprogrammed bipolar disorder patient-derived cells reveals a functional correlate of lithium responsiveness. Translational Psychiatry, 4(8), e428-.

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Metal Complex-Induced Neuroprotection

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PC12 cells were from the Cell Bank of the Chinese Academy of Sciences. Rat hippocampal neurons and neuronal medium were purchased from Sciencell (Sciencell, CA). RPMI 1640 Medium, penicillin, streptomycin and fetal bovine serum (FBS) were from Gibco. NGF-2.5S was from Life Technologies. Trypsin, sodium pyruvate and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. Reactive Oxygen Species Assay Kit was purchased from Beyotime Institute of Biotechnology. VEGF ELISA Kit was bought from Boster (Wuhan, China). The double staining apoptosis Kit was from KeyGEN (Nanjing, China). All other reagents were of analytical grade and all solutions were prepared using Milli-Q deionized water and filtered through a 0.22 micron filter. Complex 1 was synthesized in our group as reported (see Supplementary Fig. S8 online for ESI-MS spectrum)15 . Complex 2, provided by Prof. Qiuyun Chen in Jiangsu University, was synthesized as previously reported by her group21 (link). Complexes 1 and 2 were dissolved in Milli-Q deionized water as stock solutions, and diluted by culture medium to indicated concentrations.

Chen C., Cao J., Ma X., Wang X., Chen Q., Yan S., Zhao N., Geng Z, & Wang Z. (2016). Neuroprotection by Polynitrogen Manganese Complexes: Regulation of Reactive Oxygen Species-Related Pathways. Scientific Reports, 6, 20853.

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Microvascular Endothelial Cell Cultivation

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Primary human brain cortex microvascular endothelial cells [BMECs, from a single donor (24 years old, sex unknown)] were purchased from Applied Cell Biology Research Institute (Cell Systems, Kirkland, WA; catalog number ACBRI 376V). The cell line used in this study is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee. Cells were grown in endothelial growth medium 2 (EGM2; Lonza) containing endothelial basal medium 2 (EBM2; cat. No. cc-3156) supplemented with EGM2 bullet kit (cat. No. cc-3162, containing 2% fetal bovine serum, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, epidermal growth factor, ascorbic acid, hydrocortisone, bac-off and heparin). Passages 4-6 were used. In some experiments, cells were treated with human soluble APP alpha (sAPPα, Sigma-Aldrich, cat. No. s9564) 7 nM for 3 days (treatment was refreshed every 24 hours). Protein samples were then collected for Western blot.
Human neurons were purchased from ScienCell Research Laboratories (cat. No. 1520-5) and cultured in Neuronal Medium (ScienCell Research Laboratories, cat. No. 1521). The cell line used in this study is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee.

, & Upfold J. (2002). Emergency department overcrowding: ambulance diversion and the legal duty to care. CMAJ: Canadian Medical Association Journal, 166(4), 445-446.

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Cell Culture of Human Neurons and Neuroblastoma for TBEV Studies

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The virus used in this study was the TBEV strain, Neudoerfl, kindly provided by Professor F. X. Heinz from the Medical University in Vienna. The strain was originally isolated from the tick Ixodes ricinus in Austria in 1971. The virus represents a prototype strain of the European subtype of TBEV and was characterized extensively, including its genome sequence and the 3D structure of its envelope protein E42 (link). The virus underwent four passages in the brains of suckling mice before use in this study.
Human neurons (HNs) were isolated from the human brain and characterized by immunofluorescence with antibodies specific to neurofilament, microtubule associated protein 2 (MAP2), and β-tubulin III (purchased at passage zero from ScienCell Research Laboratories, Carlsbad, CA). HNs were propagated in Neuronal Medium (ScienCell) with 1% neuronal growth supplement and 1% penicillin/streptomycin (ScienCell) at 37 °C and 5% CO₂. In all experiments, cells were used at passage zero.
Human neuroblastoma cells UKF-NB-414 (link) were cultured at 37 °C and 5% CO₂ in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum (Sigma) and 1% mixture of penicillin and streptomycin (Sigma).
Porcine kidney stable (PS) cells43 (link) were cultured in L-15 medium supplemented with 3% newborn calf serum and 1% penicillin/streptomycin (Sigma-Aldrich) at 37 °C.

Bílý T., Palus M., Eyer L., Elsterová J., Vancová M, & Růžek D. (2015). Electron Tomography Analysis of Tick-Borne Encephalitis Virus Infection in Human Neurons. Scientific Reports, 5, 10745.

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Fetal Brain RNA and Neurons

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Fetal human brain RNA (#1F01-50) (Lot 1333 – Fbrain1 Male 21weeks) was purchased from Cell Applications Inc. Fetal human neuron lysate (#1526) (Lot 2219 – FNL1 sex and age not available) was purchased from ScienCell. Primary fetal neurons (#1520) (Lot 29207 – FPN1 sex unknown 19wks post-conception, Lot 28630 – FPN2 Male 22wks post conception, Lot 29390 – FPN3 Male 18wks post-conception) were purchased from ScienCell and were thawed and cultured in Neuronal Medium (ScienCell #1521). Samples were grouped by cellular age.

Capano L.S., Sato C., Ficulle E., Yu A., Horie K., Kwon J.S., Burbach K.F., Barthelemy N.R., Fox S.G., Karch C.M., Bateman R.J., Houlden H., Morimoto R.I., Holtzman D.M., Duff K.E, & Yoo A.S. (2022). Recapitulation of endogenous 4R tau expression and formation of insoluble tau in directly reprogrammed human neurons. Cell stem cell, 29(6), 918-932.e8.

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Isolation and Culture of Rat Cortical Neurons

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Cortical neurons were prepared from 1-day-old newborn rats as previously described [9 (link)]. Newborn rats were decapitated, and cerebral cortexes were transferred to PBS. After the removal of the meninges and blood vessels, tissues were cut into small pieces, followed by incubation in 0.25% trypsin-EDTA solution (Beyotime) at 37°C for 20 minutes. Then LG-DMEM with 20% fetal bovine serum was added to terminate the incubation. After centrifugation at 800 rpm for 10 minutes, the cells were collected for follow-up experiments.
Cortical neurons were dispersed with a neuronal medium (Sciencell) with 1% (vol/vol) neuronal growth supplement (Sciencell), and then they were seeded at a density of 5 × 10⁵/ml onto coverslips precoated with poly-L-lysine in 6-well plates (Corning). The medium was replaced once every 3 days, and after 7 days, the cells were fixed with 4% formaldehyde and used for the identification of neurons by immunocytochemistry.

Zhou L., Tu J., Fang G., Deng L., Gao X., Guo K., Kong J., Lv J., Guan W, & Yang C. (2018). Combining PLGA Scaffold and MSCs for Brain Tissue Engineering: A Potential Tool for Treatment of Brain Injury. Stem Cells International, 2018, 5024175.

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Differentiation of Neuronal Cell Types

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NPCs were seeded in a poly-L-lysine (P5899, Sigma, St. Louis, MO, USA) and laminin (L2020, Sigma) coated 24-well plate (1 × 10⁵ cells per well) with Neuronal Medium (1521, ScienCell, Carlsbad, CA, USA). After 10, 20, and 30 days, the immature, mature, and inhibitory neurons were observed by immunofluorescence.

Zhuang W., Liu H., He Z., Ju J., Gao Q., Shan Z, & Lei L. (2022). miR-92a-2-5p Regulates the Proliferation and Differentiation of ASD-Derived Neural Progenitor Cells. Current Issues in Molecular Biology, 44(6), 2431-2442.

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Coculture of primary cortical neurons and induced neurons

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Primary cortical neurons were isolated from P0 rat. Cortices were dissected and dissociated by trypsin digestion (0.25% Trypsin, 137 mM NaCl, 5 mM KCl, 7 mM Na₂HPO₄, 25 mM HEPES) and plated on poly-d-lysine coated glass coverslips. The neurons were maintained in growth media consisting of MEM supplemented with B27, glutamine (all from Invitrogen), glucose, transferrin (Calbiochem), fetal bovine serum and Ara-C (both from Sigma) for a week before co-culture with iNs. iNs were induced for 7 days by Induction Medium (Neuronal medium (ScienCell), 20 ng ml⁻¹ human basic Fibroblast Growth Factor, 1 mM Valproic acid) and dissociated by trypsin digestion (0.05% Trypsin). Then iN cells were seeded onto cortical neurons bed and maintained in Mature Medium (Induction Medium with 0.5 mM dbcAMP, 10 ng ml⁻¹ BDNF, 10 ng ml⁻¹ NT3). Mature Medium was replaced every 2 days.

Sun C.K., Zhou D., Zhang Z., He L., Zhang F., Wang X., Yuan J., Chen Q., Wu L.G, & Yang Q. (2014). Senescence impairs direct conversion of human somatic cells to neurons. Nature communications, 5, 4112.

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PC12 Cell Treatment Protocols

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PC12 cells and neuronal medium were obtained from ScienCell Research Laboratories (USA). Cells were cultured at 37 °C in 95% humidity and 5% CO2. 6 different treatments which cell underwent are as follows: sevoflurane (Sev), negative control oligonucleotide (NC), miR-424 mimic (mimic), pLenti-GIII-CMV vector (pcDNA), and pLenti-GIII-TLR4 plasmid (TLR4) [21 (link)].

Li Z., Wang T, & Yu Y. (2022). miR-424 inhibits apoptosis and inflammatory responses induced by sevoflurane through TLR4/MyD88/NF-κB pathway. BMC Anesthesiology, 22, 52.

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Investigating C5a-Mediated Neuronal Signaling

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Cell culture materials and reagents were from Corning (Manassas, VA, USA). Recombinant C5a and TrkA specific antagonist (AG-879) were from R&D Systems (Minneapolis, MN, USA). Anti-ß III tubulin and anti-Fibroblast Surface Protein (FSP) were from Abcam (Cambridge, UK). Anti-C5aR and anti-β-actin antibodies were respectively from Proteintech (Chicago, IL, USA) and BioLegend (San Diego, CA, USA). Fluorescent secondary antibodies were from Life Technologies (Grand Island, NY, USA), HRP-conjugated secondary antibody was from KPL (Gaithersburg, MD, USA) and IRDye secondary antibodies were from LI-COR (Lincoln, NE, USA). Low-fluorescence PVDF transfer membranes were from Thermo Scientific (Waltham, MA, USA). The C5aR antagonist (W54011), and chambers to evaluate the neurite outgrowth (AXIS) were from EMD Millipore (Darmstadt, Germany). The human beta NGF ELISA kit and lipoteichoic acid (LTA) were from Sigma-Aldrich (St. Louis, MO, USA), and chemicals were from Fisher Chemical (Nazareth, PA, USA). Human neurons, neuronal medium and neuronal growth supplement were from Sciencell (Carlsbad, CA, USA). All experimental protocols used for this study were in accordance with the guidelines according to the Institutional Animal Care and Use Policy and approved by the IRB Protocol Committee at the University of Illinois at Chicago.

Chmilewsky F., Ayaz W., Appiah J., About I, & Chung S.H. (2016). Nerve Growth Factor Secretion From Pulp Fibroblasts is Modulated by Complement C5a Receptor and Implied in Neurite Outgrowth. Scientific Reports, 6, 31799.

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