Dmem f12 neurobasal

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DMEM:F12/Neurobasal is a basal cell culture medium developed for the maintenance and growth of various cell types, including neuronal and stem cells. It is a combination of two widely used media formulations, DMEM:F12 and Neurobasal, which are designed to provide a balanced nutrient environment for optimal cell proliferation and differentiation.

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24 protocols using dmem f12 neurobasal

Culturing Mouse and Human Neural Progenitor Cells

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mESCs (E14TG2a.4, ATCC) were cultured on gelatine in 2i medium: 50:50 DMEM/F12:Neurobasal (Invitrogen) supplemented with N2/B27 (Invitrogen), 0.05 mM β-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM GlutaMAX, 1 mM sodium pyruvate, Pen/Strep, leukemia inhibitory factor (LIF), 1 μM MEK inhibitor (PD0325901) and 3 μM GSK inhibitor (CT-99021).
Mouse neural progenitor cells derived from mESCs were cultured on PDL/Laminin in complete NSC medium: 50:50 DMEM/F12:Neurobasal (Invitrogen) supplemented with N2/B27 (Invitrogen), 0.05 mM β-mercaptoethanol, 2 mM GlutaMAX, 0.1 mM non-essential amino acids, Pen/Strep, 1 mM sodium pyruvate, 5mM Hepes, 2 µg/ml Heparin, 50 µg/ml BSA, 10 ng/ml bFGF/EGF
Human GNS line G144 (19 (link)) and human fetal neural stem cell lines 18.5 and 21.5 (a kind gift of Dr. Steven Goldman, University of Rochester Medical Center, USA) were cultured on PDL/Laminin in complete NSC medium.
Flp-In-T-REX-293 cells were cultured in DMEM (Invitrogen) with 10% FBS (HyClone) and Pen/Strep. Cells were authenticated by measuring the expression of specific cellular markers and by observing their morphology and functionality in specific assays. All the cell lines used were routinely (every 4 weeks) tested negative for mycoplasma by PCR. Cell culture time between thawing and experiment did not exceed 5 passages.

Weissmann S., Cloos P.A., Sidoli S., Jensen O.N., Pollard S, & Helin K. (2018). The tumor suppressor CIC directly regulates MAPK pathway genes via histone deacetylation. Cancer research, 78(15), 4114-4125.

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NCSC Migration in Hyaluronic Acid Hydrogel

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We performed the following set of experiments in order to study the migration of NCSCs in hyaluronic acid hydrogel (Healon 5®). Plain NCSC neurospheres were applied onto the biomaterial and maintained for 3, 6 and 10 days in the presence of NCSC-differentiation medium consisting of 1:1 DMEM/F12/Neurobasal supplemented with 1.2% non-essential amino acids (NEAA, 100×), 1% N2 (100×) and 1% B27 (50×; all from Invitrogen). Neurospheres maintained on standard laminin-coated coverslips in the presence of NCSC-differentiation medium were used as controls. Migration of NCSCs was studied using light and fluorescent microscopy.

Schizas N., König N., Andersson B., Vasylovska S., Hoeber J., Kozlova E.N, & Hailer N.P. (2018). Neural crest stem cells protect spinal cord neurons from excitotoxic damage and inhibit glial activation by secretion of brain-derived neurotrophic factor. Cell and Tissue Research, 372(3), 493-505.

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Direct Neuronal Conversion of Glioma Cells

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Human glioma cell lines, U251 and U87, were cultured in DMEM supplemented with 10% fetal bovine serum. Cells were seeded on gelatin- or Matrigel-coated culture vessels with or without glass coverslips. The following day, they were infected with the indicated lentivirus in the presence of 6 μg/ml polybrene. After overnight incubation, culture media were refreshed. One day later, the cells were then switched to neuronal induction medium, which consists of DMEM:F12:Neurobasal (2 : 2 : 1), 0.8% N-2 (Invitrogen, Carlsbad, CA, USA), and 0.4% B-27 (Invitrogen). In addition, forskolin (FSK, 10 μM) and dorsomorphin (DM, 1 μM) were added to the above induction medium. Induction medium was half-changed every other day. TUJ1⁺ cells with a neuronal morphology, indicated by a round or pyramidal soma with a thin process at least three times longer than the cell body, were counted at the indicated time points. The conversion efficiency was estimated by dividing the total number of neuron-like cells by the number of virus-infected cells (indicated by GFP) or initially seeded cells.

Su Z., Zang T., Liu M.L., Wang L.L., Niu W, & Zhang C.L. (2014). Reprogramming the fate of human glioma cells to impede brain tumor development. Cell Death & Disease, 5(10), e1463-.

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Astrocyte-to-Neuron Reprogramming Protocol

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Spinal astrocytes from the mGfap-Cre;Rosa-tdT mice were purified through fluorescence activated cell sorting based on tdT expression. These astrocytes were then infected with the hGFAP-GFP-T2A-SOX2 or hGFAP-GFP lentivirus. Three days later, the culture was switched to NSC medium, which consists of DMEM:F12:Neurobasal (2:2:1), 0.8% N2, 0.4% B27 (Life Technologies), 20 ng/ml FGF2, and 20 ng/ml EGF (Peprotech). Primary neurospheres were collected at day 14 and serially passaged under the same culture condition. To induce differentiation, secondary neurospheres were dissociated into single cells, which were then seeded onto Matrigel-coated glass coverslips in 24-well plate at a density of 2 × 10⁴ cells per well. Cells were further cultured for 6 days in the NSC medium without FGF2 and EGF. Immunocytochemistry was performed on 4% paraformaldehyde-fixed cells using the indicated primary antibodies.

Su Z., Niu W., Liu M.L., Zou Y, & Zhang C.L. (2014). In vivo conversion of astrocytes to neurons in the injured adult spinal cord. Nature communications, 5, 3338.

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Astrocyte-to-Neuron Reprogramming Protocol

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Su Z., Niu W., Liu M.L., Zou Y, & Zhang C.L. (2014). In vivo conversion of astrocytes to neurons in the injured adult spinal cord. Nature communications, 5, 3338.

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Directed Differentiation of mESCs to NPCs

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ES to NPC differentiation was performed according to standard protocols with small modifications as previously described (Conti et al, 2005 (link); Splinter et al, 2011 (link); Bowness et al, 2022 (link)). First, mESCs were extensively isolated from feeder cells by four rounds of pre-plating, each for 35–40 min. 0.5 × 10⁶ mESCs were plated to gelatin-coated T25 flasks in N2B27 media (50:50 DMEM/F-12:Neurobasal (Gibco) supplemented with 1× N2 and 1× B27 (ThermoFisher), 1 mM l-glutamine, 100 μM β-mercaptoethanol, 50 U/mL penicillin/50 μg/mL streptomycin (all from Life Technologies) with 1 μg/ml doxycycline for continuous Xist induction. Media changes were performed every day except day 1. After 7 days, cells were detached with Accutase (Merck Life Sciences) and 3 × 10⁶ cells were plated to grow in suspension in 90 mm bacterial petri dishes containing N2B27 media supplemented with 1 μg/ml doxycycline and 10 ng/ml EGF and FGF (Peprotech). Embryoid-body-like aggregates were collected by mild centrifugation (100× g for 2 min) on day 10 and re-plated to 90 mm gelatinised cell culture dishes in N2B27 + dox + FGF/EGF media, with media changes performed every other day. Upon reaching ~80% confluency, NPC outgrowths were passaged using Accutase at a 1:3–1:4 ratio to new cell culture dishes until the establishment of a homogenous cellular population of NPCs from approximately day 15–17.

Bowness J.S., Almeida M., Nesterova T.B, & Brockdorff N. (2024). YY1 binding is a gene-intrinsic barrier to Xist-mediated gene silencing. EMBO Reports, 25(5), 2258-2277.

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Generation of Neurons from Human iPSCs

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Human iPS cell line (Foreskin-1, WiCell24 (link)) was used for generation of neurons out of primitive neural stem cells (pNSCs). Therefore, iPS cells were cultured on feeder cells to form small colonies. Next, medium was changed to neural stem cell medium (DMEM/F12: Neurobasal; GIBCO) in the presence of leukaemia inhibiting factor (LIF; Millipore, 10 ng/ml) and three small molecules CHIR99021 (inhibitor of GSK-3β, Axon Medchem, 4 μM) and SB431542 (inhibitor of TGF-β and activin receptors; Axon Medchem, 3 μM), and Compound E (inhibitor of γ-secretase; Axon Medchem, 0.1 μM) for 10 days. To induce differentiation towards neurons, pNSCs were dissociated by accutase (PAA) and cultured on poly-L-ornithine (Sigma, 0.15 mg/ml) plus laminin (Sigma, 1 μg/ml) coated cell culture dishes in neural stem cell medium (DMEM/F12:Neurobasal, plus LIF, CHIR99021 and SB431542) till cell attached and formed small colonies. Then, medium was changed to neuronal differentiation medium (DMEM/F12, plus N2 and B27 supplements, GIBCO) plus ascorbic acid (TOCRIS, 0.2 mM) and cyclic adenosine monophosphate (Sigma, 300 ng/ml) in presence of brain derived neurotrophic factor (BDNF; Prospect, 10 ng/ml) and glial cell line-derived neurotrophic factor (GDNF; Prospect, 10 ng/ml) for 2 weeks. Medium containing the neurotrophic factors was changed every second day.

Shahraz A., Kopatz J., Mathy R., Kappler J., Winter D., Kapoor S., Schütza V., Scheper T., Gieselmann V, & Neumann H. (2015). Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages. Scientific Reports, 5, 16800.

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3D Neural Differentiation of iPSC-NPCs

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For 3D neural differentiation of iPSC–NPCs, normal‐ or PS1‐mutated AD‐iPSCs‐NPCs were seeded at a density of 5 × 10⁴ cells in a U‐bottom plate (Thermo Fisher Scientific, Waltham, MA) and then placed in NPC media for 3 days. The medium was changed to neuronal differentiation medium (NDM) on the following day and then every 2 days for 1 week. NDM comprised a 1:1 mixture of DMEM F12: Neurobasal (GIBCO) supplemented with 1× Glutamax (GIBCO) and 1% B27 supplement without vitamin A (GIBCO). Cell culture medium was changed to glial differentiation medium (GDM) on the following day and then every 2 days for 2 weeks. GDM comprised ScienCell astrocyte medium (Cat. 1801, San Diego, CA) supplemented with 1:100 antimycotic‐antibiotics, 1:50 fetal bovine serum, and astrocyte growth supplement (ScienCell Cat. 1852). Spheroids were fixed or subjected to drug efficacy testing at 21 days post differentiation.

Park H., Kim J, & Ryou C. (2023). A three‐dimensional spheroid co‐culture system of neurons and astrocytes derived from Alzheimer's disease patients for drug efficacy testing. Cell Proliferation, 56(6), e13399.

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Cerebral Organoid Differentiation from hESCs

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Differentiation of hESCs into cerebral organoids was based on a previously described protocol^{26 (link),29 (link)}. Briefly, hESC cells were inoculated into a spinner flask, and to enable embryoid body formation, after six days the medium was changed to neural induction media (DMEM/F12, 1X N2 supplement (Gibco), 2 mM Glutamax (Invitrogen), 1% MEM-NEAA, and 1 μg/mL heparin (Sigma)) and the aggregates were cultured for five more days. After being embedded in matrigel, differentiation media composed of 1:1 DMEM/F12:Neurobasal (Gibco), 0.5X N2, 1X B27 minus vitamin A (Gibco), 2 mM Glutamax, 0.5% MEM-NEAA, 0.2 μM 2-mercaptoethanol and 2.5 μg/mL insulin was used. After 4 days, cell aggregates were grown in neuronal differentiation media, composed as aforementioned except by replacing with 1X B27 containing vitamin A (Gibco). The medium was changed once per week. Cerebral organoids were grown for 45 days (30 days in neuronal differentiation media).

Dakic V., Minardi Nascimento J., Costa Sartore R., Maciel R.D., de Araujo D.B., Ribeiro S., Martins-de-Souza D, & Rehen S.K. (2017). Short term changes in the proteome of human cerebral organoids induced by 5-MeO-DMT. Scientific Reports, 7, 12863.

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Retroviral Lineage Tracing of Human NSCs

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Replication-incompetent retrovirus was produced as described in supplementary methods. For retroviral lineage-tracing experiments, human NSCs were plated at a density of 5×10⁴ cells/cm² onto coverslips or dishes coated with poly-ornithine (10 μg/ml, Life Technologies) and laminin (5 μg/ml, Life Technologies). The next day, the medium was changed to neuronal differentiation medium (NDM) and sparse labeling of NSCs was achieved by using short time pulse infections with low-titer retroviral particles (for constructs, see supplementary methods). NDM is based on DMEM:F12/Neurobasal (both Gibco) (1:1) and contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco) and laminin (1 μg/ml, Life Technologies). After six hrs, the remaining viral particles were washed out and the medium was replaced with NDM. For subsequent analyses, maturing neuronal cultures were followed for 2, 4, 7 and 14 dpi in vitro before being processed for immunocytochemistry, electrophysiology, flow cytometry or FACS-based purification.

Schafer S.T., Paquola A.C., Stern S., Gosselin D., Ku M., Pena M., Kuret T.J., Liyanage M., Mansour A.A., Jaeger B.N., Marchetto M.C., Glass C.K., Mertens J, & Gage F.H. (2019). Pathological priming causes developmental gene network heterochronicity in autism patient-derived neurons. Nature neuroscience, 22(2), 243-255.

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