At Day 28 of the culture, neurosphere media were changed to Neurobasal Medium minus phenol red (Cat# 12348‐017, Gibco) supplemented with B‐27 Supplement Minus AO (Cat# 10889038, Gibco) and 2 mM l ‐glutamine. ZNS was added at three different concentrations: 0.05, 0.5, and 5 µM. After 1 day of ZNS exposure, the cells were incubated with 1 mM H2O2 for 12 hr. The viability of the cells was examined with an LDH Cytotoxicity Detection Kit (Cat# MK401, Takara Bio, Shiga, Japan) according to the manufacturer's protocol.
Neurobasal medium minus phenol red
Manufactured by Thermo Fisher Scientific
Neurobasal medium minus phenol red is a serum-free, defined medium formulation optimized for the culture of primary neurons and other central nervous system-derived cells. It is a modification of the original Neurobasal medium, with the removal of the pH indicator phenol red.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
Hfgf2, by R&D Systems (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
B 27 supplement minus ao, by Thermo Fisher Scientific (1 mentions)
Ldh cytotoxicity detection kit, by Takara Bio (1 mentions)
Human epidermal growth factor (hegf), by R&D Systems (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Accutase, by STEMCELL (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
12 well plate, by Corning (1 mentions)
Fv10 asw, by Olympus (1 mentions)
Magnetic bead sorting, by Miltenyi Biotec (1 mentions)
Papain, by Worthington (1 mentions)
6 protocols using neurobasal medium minus phenol red
1
Neuroprotective Effects of ZNS
2
Culture Conditions for Brain Cell Lines
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All cells were grown at 37°C with 5% CO2. The L0, L1, L2, DI318, G3691, G3832, and GSC23 lines were grown in Neurobasal medium minus phenol red (Gibco) with 1X B-27 supplement (Gibco), 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 50 U/mL penicillin/streptomycin, 20 ng/ml hEGF and 20 ng/ml hFGF2 (R&D Systems). The remaining cell lines were grown in DMEM-F12 (Cleveland Clinic Media Preparation Core), 50 U/mL penicillin/streptomycin, 1% N2 supplement (Thermo Fisher Scientific), 20ng/ml FGF-2 (R&D Systems). Cells were passaged regularly using Accutase (Stem Cell Technologies) and phosphate-buffered saline.
3
Microfluidic Culture of Hippocampal Neurons
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Hippocampal neurons were isolated by standard methods using C57BL/6 mice sacrificed at E17 and grown in culture chamber microfluidic devices (Xona Microfluidics) placed on 24 × 60 mm coverslips (#1,5 Menzel-Glaser) that had been coated with poly-D-lysine (PDL), to form a non-plasma bond with the device. 60,000–80,000 neurons were plated per chamber using Neurobasal medium (21,103,049, Thermo-Fisher) supplemented with 5% fetal bovine serum (FBS; Hyclone), 2% B27 (17,504,044, Thermo-Fisher), 1 mM GlutaMAX (35,050,061, Thermo-Fisher) and 50 U/ml penicillin/streptomycin (15,070,063, Thermo-Fisher). The medium was changed to serum-free Neurobasal medium minus phenol red (12,348,017, Thermo-Fisher), supplemented with 28 nM 2-mercaptoethanol (21,985,023, Thermo-Fisher) 24 h post-seeding, and half of the medium was changed twice a week. A hydrostatic pressure gradient that prevents diffusion between culture chamber (Ch) 1 and Ch2 was established by adding twice the volume of culture medium to Ch2. To perform electron microscopy, the microfluidic devices were placed on plastic culture dishes coated with PDL. All cultures were maintained at 37 °C and 5% CO2 for up to 12 days. Neurons grown in the microfluidic devices were analyzed at 8–12 days in vitro (DIV8–12).
4
Culturing Hippocampal Neurons from Mice
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Hippocampal neurons were established from C57BL/6 mice at E17 and grown on 18-mm coverslips coated with poly-D-lysine (PDL) placed in 12-well plates (Corning) [32 (link)]. Neurons were plated at a density of 60,000 neurons per well for imaging or 300,000 neurons for western blots, using Neurobasal medium (21103049, Thermo-Fisher) supplemented with 5% serum (FBS; Hyclone), 2% B27 (17504044, Thermo-Fisher), 1 mM GlutaMAX (35050061, Thermo-Fisher), and 50-U/ml penicillin/streptomycin (15070063, Thermo-Fisher). The medium was changed to serum-free Neurobasal medium minus phenol red (12348017, Thermo-Fisher), supplemented with 28-nM 2-mercaptoethanol (21985023, Thermo-Fisher), and 25-µM glutamic acid 24 h post-seeding, and half of the medium was changed twice a week [8 (link), 80 (link)]. All cultures were maintained at 37 °C and 5% CO2 for up to 10 days in vitro (DIV10). For some experiments, neurons were transduced with lentivirus at DIV2, then treated with exosomes at DIV7 by taking out 500 µl of the neuron-conditioned medium and replacing it with 500-µl fresh Neurobasal culture medium containing 10-µg protein equivalents of exosomes for neurons to be imaged or 20 µg for neurons to be analyzed by western blotting, added dropwise and in the absence of lipofectamine.
5
In Utero Midbrain Slice Culturing
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Two days after in utero electroporation, E15.5 midbrains were embedded in 3% low-gelling temperature agarose (A9045-25G; Sigma-Aldrich) in Hanks’ balanced salt solution (HBSS), after which 250-μm coronal slices were obtained with a vibratome (VTI1000S; Leica). E15.5 midbrain slices were placed on a Millicell-CM (PICM0RG50; Millipore, Burlington, MA), mounted in 70% collagen gel (KP-2100; Nitta Gelatin, Fayetteville, NC), and soaked in culture medium containing Neurobasal medium minus phenol red (12348017; Thermo Fisher Scientific), 1× B27 supplement (17504044; Invitrogen), and 1 mM l -glutamine in a glass-bottom dish (3910-035; Iwaki, Tokyo, Japan). The slices in Millicell-CM dishes were kept at 37°C in a CO2 incubator (Olympus, Tokyo, Japan) equipped with a motorized inverted research microscope with focus-drift compensation IX81 (Olympus) and set at the heat stage. Neurons were observed under 5% CO2 and 60% O2. Time-lapse recordings were performed by epifluorescence microscopy with a 20× long operation distance objective lens (LMPLFLN-BD 20×, 0.40 numerical aperture; Olympus). Image acquisition was performed with Dell computers using an FV10-ASW (Olympus) control camera. The time interval of time-lapse recording was 10 min for all movies. The medium intensity projection was prepared from 10 to 15 Z-stack images at each time point.
6
Derivation and Culture of GBM Tumor Cells
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GBM Tumor cell derivation and culture GBM tumor models were generated by passaging primary tumor cells through immunocompromised mice as previously described (Bao et al., 2006b; Lathia et al., 2010) (link). Briefly, primary tumor cells were intracranially implanted into NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice, and upon tumor formation, tumors were isolated and digested with papain (Worthington). Dissociated cells were plated overnight in Neurobasal™ Medium minus phenol red (ThermoFisher) with 1x B27 supplement (ThermoFisher), 1 mM sodium pyruvate, 2 mM Lglutamine, 50 U/mL penicillin/streptomycin, 20 ng/ml human (h)EGF and 20 ng/ml hFGF2 (R&D systems). Subsequently, CD133+ cells were isolated by magnetic bead sorting (Miltenyi) and cultured in the media described above. Some cell models were previously established at Duke University and obtained through approved material transfer agreements. CD133+ cells were seeded in suspension culture at 5x10 4 cells/ml and passaged no more than 10 times. After 10 passages, cells were re-implanted into NSG mice and enriched for CD133+ cells. De-identified GBM specimens were collected from the Cleveland Clinic Brain Tumor and Neuro-Oncology Center in accordance with an Institutional Review Board-approved protocol, and informed consent was obtained from all GBM patients contributing tumor specimens.
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