After microglia removal, OPCs growing on top of astrocyte monolayer were isolated by shaking cells on an orbital shaker at 200 rpm for 3 h and incubated on an uncoated Petri dish for 1 h to further eliminate microglia. Pure OPCs (> 95% [32 (link)] were seeded onto poly-d ,l -ornithine-coated glass coverslips or plates (50 μg ml−1, Sigma-Aldrich, Milan, Italy) in Neurobasal (Life Technologies, Monza, Italy) supplemented with 2% B27 (Life Technologies, Monza, Italy), 2 mM l -glutamine (EuroClone, Milan, Italy), 10 ng ml−1 human platelet-derived growth factor BB (Sigma-Aldrich, Milan, Italy), and 10 ng ml−1 human basic fibroblast growth factor (Space Import Export, Milan, Italy), to promote proliferation (proliferating medium). After 3 days, cells were either detached with accutase (Millipore, Burlington, MA, USA) and used for migration assay, or switched to a Neurobasal medium lacking growth factors and supplemented with triiodothyronine T3 (10 ng ml−1, Sigma Aldrich, Milan, Italy) to allow differentiation (differentiating medium).
Neurobasal
Manufactured by Merck Group
Sourced in United States
Neurobasal is a cell culture medium designed for the maintenance and growth of primary neuronal cells. It provides a specialized formulation that supports the survival and differentiation of these delicate cell types. The core function of Neurobasal is to create an optimized environment for culturing neurons and maintaining their viability in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal, by Thermo Fisher Scientific (6 mentions)
Accutase, by Merck Group (2 mentions)
Triiodothyronine t3, by Merck Group (2 mentions)
L glutamine, by Euroclone (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Glutamine, by Merck Group (2 mentions)
Human platelet derived growth factor bb, by Merck Group (1 mentions)
Glass coverslip, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Noggin, by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 fgf2, by Thermo Fisher Scientific (1 mentions)
Fluozin 3 am, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s media dmem, by Merck Group (1 mentions)
Horse serum, by Atlanta Biologicals (1 mentions)
12 protocols using neurobasal
1
Isolation and Differentiation of Oligodendrocyte Progenitors
2
Preparation of Hippocampal Primary Cultures
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Hippocampal primary cultures were prepared from embryonic 20-day old Sprague-Dawley rats. The embryonic rats were removed from deeply anesthetized pregnant rats, then transferred to an ice-cold normal Tyrode solution containing the following (in mM): 140 NaCl, 5.4 KCl, 2.3 MgCl2, 10 HEPES, 5 glucose, pH 7.4 adjusted with NaOH. Isolation of the hippocampi from embryonic rat brains was performed in a chamber containing ice-cold normal Tyrode solution under a microscope in a sterilized environment. Dissected hippocampi were transferred to ice-cold minimal essential medium (MEM) containing Earle's salts and glutamine with 10% fetal bovine serum, 0.45% glucose, 1 mM sodium pyruvate, 25 µM glutamate and antibiotics, and then triturated using 200 µL pipettes. The cells were counted and seeded on 12 mm-diameter glass cover slips (Fisher Scientific) coated with poly-L-lysine (Sigma-Aldrich) at a density of 9×104 cells/mL and maintained at 37℃ in 95% air and 5% CO2. After 7 hours, the whole plating medium was changed to Neurobasal (Sigma-Aldrich) medium containing B-27 (Invitrogen), and half of the medium was changed once at DIV 4. All experiments and procedures with animals were performed with the permission from the Animal Care and Use Committee of Jeju National University.
3
Differentiation of iPSCs to Cortical Neurons
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The differentiation of iPSCs to CNs was performed following a previously established protocol 34 . Briefly, the iPSCs were grown on Matrigel as monolayers, and neural differentiation was induced by changing the medium to basic neural medium DMEM/F12: Neurobasal 1:1, 1× N2, 1× B27, insulin 5 µg/mL (Sigma), nonessential amino acids 100 µM (Life Technologies), 2‐mercaptoethanol 100 µM, antibiotic‐antimycotic (Life Technologies) and supplementing it with 500 ng/mL Noggin (PeproTech, Rocky Hill, NJ, http://www.peprotech.com ), and 10 µM SB431542 (Enzo Life Sciences). Medium was changed every 24 hours until neural rosettes could be observed (12–16 days). The neuroepithelial layer was lifted using Dispase (Life Technologies), and the medium was supplemented with 20 ng/mL Fibroblast growth factor 2 (FGF2) (PeproTech) for 4–6 days. The neural precursors were split using Accutase (Life Technologies) at a low density. The medium was then supplemented with 10 ng/mL BDNF and 10 ng/mL GDNF, while FGF2 was withdrawn. The neurons were allowed to extend processes and to mature for 60–70 days before functional assays.
4
Neuronal Cell Culture Reagents
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Cell culture reagents were purchased from Life Technologies [Trypsin-EDTA (0.05%), TrypLE, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), Anti-Anti, B-27 supplement along with Neurobasal, Dulbecco’s modified Eagle’s media (DMEM) and Minimum Essential Media (MEM) medias], Sigma-Aldrich [Cytosine β-D-arabinofuranoside (Ara-C), poly-D-lysine (70–150 kDa), Hank’s Balanced Salt Solution (HBSS), 1,4-Piperazinediethanesulfonic acid (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), insulin, N-Acetyl Cysteine (NAC), hydrocortisone, sodium pyruvate and GlutaMAX], Worthington (Trypsin, Papain, Papain Dissociation Kit, and DNase), Atlanta Biologicals [Horse Serum (HS) and Fetal Bovine Serum (FBS)]. Zinc indicators (FluoZin-3 AM and Newport Green DCF) were obtained from Life Technologies. Vitamin MEM solution, amino acid MEM solution, ZnCl2 (0.1 M stock solution), N,N,N′,N′-Tetrakis (2-pyridylmethyl)ethylenediamine (TPEN), 2-Mercaptopyridine N-oxide sodium salt (pyrithione) were all purchased from Sigma-Aldrich. For GluA2 surface labeling, Alexa 488-CAM2 and Alexa 647-CAM2 were designed and synthesized by the Hamachi research group (Kyoto University; Wakayama et al., 2017 (link)).
5
Cortical Neuron Dissection and Culture
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Cortices were micro-dissected from 5 SAS Sprague Dawley Rat embryos or 7 to 10 E14 embryos of PS19 mice. All steps of the dissection were performed in cold Gey’s balanced salt solution (GBSS) supplemented with 0.1% glucose. Dissected structures were digested with papain (20U/ML in Neurobasal, Sigma). After papain inactivation with FBS, structures were mechanically dissociated with a pipette in presence of DNAse. After several rounds of rinsing, cells were re-suspended in Neurobasal + B27 in a final density of 35 million cells/mL. For 24 well plate, 150 000 to 200 000 of cortical precursors are plated. For microfluidic cultures, WT cortical neurons were then seeded in the left and the right chamber: 2 µl of the cell suspension was introduced into the upper reservoir and cells flowed into the chamber and adhered within 20 min. For 24 well plate and for microfluidic devices, cortical neurons are plated in plating media for 24 h: Neurobasal + B27 (Gibco) + streptomycin/penicillin (Gibco) + 10% FBS. The next day, a media change is performed to remove the FBS, Neurobasal + B27 (Gibco) + streptomycin/penicillin (Gibco). Microfluidic chips were placed in plastic Petri dishes containing H2O-EDTA to prevent evaporation and incubated at 37 °C in a humid 5% C02 atmosphere. The culture medium was renewed every 6 days.
6
Visualizing GluR1 and GluR2 in Hippocampal Neurons
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At DIV13-20 hippocampal neurons were incubated with GluR1 (Calbiochem (1:8) and GluR2 (Zymed (1:80) amino-terminal antibodies (10 µg/mL)) at 37°C for 15 min (Martin et al. 2008 (link)). Cells were preincubated at 37°C in 5% CO2 for 1 h in Neurobasal containing the potent mTOR inhibitor rapamycin (50 nM, Sigma) followed by corticosterone (100 nM, Sigma) or vehicle for 3 h in the presence of rapamycin. After washing in DMEM medium, the neurons were fixed for 5 min with 4% formaldehyde/4% sucrose in phosphate-buffered saline (PBS). Neurons were then washed three times in PBS for 30 min at room temperature and incubated with secondary antibody conjugated to Alexa488 (1:400) or Alexa568 (1:400) in staining buffer without TritonX-100 (0.2% BSA, 0.8 M NaCl, 30 mM phosphate buffer, pH 7.4) overnight at 4°C. Neurons were then washed three times in PBS for 30 min at room temperature and mounted. Confocal images were obtained with sequential acquisition settings at the maximal resolution of the microscope (1024 × 1024 pixels). Morphometric analysis and quantification were performed using MetaMorph software (Universal Imaging Corporation).
7
Multilineage Differentiation of EB-NPCs
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For flow cytometric analysis of EB-NPCs, cells were collected using Accutase and stained with mouse anti-Nestin, mouse anti-human Sox1, and mouse anti-Sox2 per manufacturer’s instructions using the Human Neural Lineage Analysis kit (BD). For immunofluorescence microscopy, EB-NPCs were seeded on Geltrex-coated slides, fixed with 4% paraformaldehyde, and stained with rat anti-Nestin (1:500; Millipore), rabbit anti-Sox2 (1:200; Epitomics), or rabbit anti-Pax 6 (1:50; BioLegend) before addition of respective secondary antibodies (goat anti-rabbit AlexaFluor 568 and goat anti-rat AlexaFluor 488; both from Thermo Fisher). For analysis of multipotency, EB-NPCs were cultured in neuronal differentiation medium (Neurobasal, 1X B27, 1X GlutaMAX), astrocyte differentiation medium (DMEM [Thermo Fisher], 1X N2, 1X GlutaMAX, 1% FBS [Atlanta Biologicals]), or oligodendrocyte differentiation medium (Neurobasal, 1X B27, GlutaMAX, 30 ng/ml T3 [Sigma]) for at least 14 days before being fixed with 4% paraformaldehyde and stained with mouse anti-beta-III-tubulin (1:500; Abcam), rabbit anti-GFAP (1:200; Thermo Fisher), rabbit anti-NG2 (1:200; Chemicon), or rabbit anti-Olig2 (1:100; Abcam). Slides were cover-slipped and mounted using VectaShield Hard Set Mounting Medium with DAPI (Vector Labs), and all images were captured using a Nikon Eclipse Ti inverted microscope.
8
Isolation and culture of rat OPCs
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We isolated OPCs from rat (Sprague-Dawley) cerebral cortex at postnatal day 2 (P2) by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) utilizing a Papain-based Neural Tissue Dissociation Kit (Miltenyi Biotec, Bologna, Italy) and anti-A2B5 magnetic microbeads (Miltenyi Biotec, Bologna, Italy). The cells were plated onto poly-d,l-ornithine-coated glass coverslips in Neurobasal (Life Technologies, Monza, Italy) supplemented with 2% B27 (Life Technologies, Monza, Italy), l-glutamine (2 mM; EuroClone, Milan, Italy), human platelet-derived growth factor (PDGFBB, 10 ng/ml; Sigma-Aldrich, Milan, Italy), and human basic fibroblast growth factor (bFGF, 10 ng/ml; Space Import Export, Milan, Italy), to promote proliferation (proliferating medium). 3 days after, we detached OPCs with accutase (Millipore, Burlington, MA, USA) for migration analysis or incubated the cells with a differentiating medium, i.e. Neurobasal medium devoid of growth factors and containing triiodothyronine T3 (10 ng/ml; Sigma Aldrich, Milan, Italy).
9
Neuronal Differentiation from Dissociated Cells
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Dissociated cells were reseeded onto laminin coated plates in differentiation medium [Neurobasal with 1% N2, 2% B27, 1% NEAA supplemented with 10 ng/ml BDNF, 10 ng/ml GDNF, and 200 μM ascorbic acid (all from Sigma-Aldrich), without growth factors- bFGF and EGF] for up to 3–4 weeks and differentiated spontaneously. Half the volume of the medium was replaced by fresh medium every 2–3 days. For spheroid differentiation, cells were trypsinized into single cells and transferred into non-adherent plates in EB medium without any anti-differentiation factors.
10
Culturing Primary Mesencephalic Neurons
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Pregnant mice at 13–16 days were sacrificed for mesencephalic primary neurons culture. Fetal mice were washed with sterile PBS containing penicillin and streptomycin for three times, and placed in high‐glucose DMEM. The midbrain was isolated under sterile conditions with a microscope, the meninges and blood vessels were gingerly removed, 0.25% trypsin was added and digested in 37°C water bath for 8 min. Added DMEM medium containing 10% FBS and 10% horse serum, cells was shaken into single‐cell suspension, which was filtered by a 200‐mesh filter. After 24 h, the medium was half‐replaced with Neurobasal (Gibco, USA) containing 2% B27 (Gibco, USA) and 25 μM glutamine (Sigma, USA), then added with 1 μM cytarabine (Sigma, USA) after 24 h. In the next 24 h, the whole medium was replaced with Neurobasal containing 2% B27 and 25 μM glutamine. After three days of culture, the purity of neurons was higher than 95% by MAP2 (neuron maker) staining, then collected cells for the following experiments.
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