Primary mouse cortical neurons were prepared as previously described [11] (link), [14] (link). Briefly, cerebral cortices were isolated from C57BL/6J mouse embryos on the 17th embryonic day, minced and treated with dissociation solution (Sumitomo Bakelite, Akita, Japan). Neurons were resuspended in Nerve Culture Medium (Sumitomo Bakelite), plated on polyethylenimine-coated glass coverslips (Asahi Techno Glass, Chiba, Japan) at a density of 5×103 cells/well in 96-well multidishes, 5×104 cells/well in 24-well multidishes, or 6×106 cells/well in 6-well multidishes, and incubated at 37°C in an atmosphere containing 5% CO2 at 100% humidity. The purity of the cultures was greater than 95% based on NeuN-specific immunostaining. Neurons were used at 14 days in vitro for the following assessments.
Dissociation solution
Manufactured by Sumitomo Bakelite
Sourced in Japan
Dissociation solution is a laboratory product used to facilitate the separation of cells or tissues during various experimental procedures. The solution contains a combination of chemical agents that disrupt the cellular adhesion, allowing for the isolation and extraction of individual cells or cellular components. This product serves as a core tool in cell biology, tissue engineering, and related fields of study, providing a standardized method for sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nerve culture medium, by Sumitomo Bakelite (3 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Neuron culture medium, by Sumitomo Bakelite (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
24 well plate inserts, by Corning (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
5 protocols using dissociation solution
1
Primary Mouse Cortical Neuron Isolation
2
Preparation of Primary Rat Cortical Neurons
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Primary neuron cultures were prepared from cortices of embryonic day 17 (E17) Wistar rat embryos. Briefly, cortical fragments were dissociated into single cells in dissociation solution (Sumitomo Bakelite; Akita, Japan) and resuspended in nerve culture medium (Sumitomo Bakelite). Neurons were plated onto 12-mm polyethyleneimine (PEI)-coated glass coverslips (Asahi Techno Glass; Chiba, Japan) at a density of 5 × 104 cells/well in 24-well plates and incubated at 37 °C in a humidified atmosphere containing 95% O2/5% CO2. The purity of the cultures was > 95%, as determined via neuronal nuclei (NeuN)-specific immunostaining.
3
Primary Neuronal Culture and BBB Model
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Primary neuronal cultures were prepared from the cortices of C57BL/6 mouse embryos at embryonic Day 17 as described previously [20] (link). Briefly, cortical fragments were dissociated into single cells in dissociation solution (Sumitomo Bakelite, Akita, Japan), and resuspended in neuron culture medium (Sumitomo Bakelite). Neurons were seeded onto 12-mm polyethylenimine-coated glass coverslips (Asahi Techno Glass Corp., Chiba, Japan) at a density of 5.0×104 cells/well in 24-well culture plates and were incubated at 37°C in a humidified atmosphere containing 5% CO2. The purity of the cultures was >95% as determined by NeuN-specific immunostaining. Mouse brain capillary endothelial cell line MBEC4 [21] (link) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Confluent monolayer of MBEC4 cells was used as an established BBB model as described previously [22] (link).
4
Neuroblastoma Cell Co-culture System
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Neuroblastoma SH-SY5Y cells with and without stable transfection of human APP770 were maintained in Eagle’s minimum essential medium/Ham’s F-12 medium (Thermo Scientific, Waltham, MA) supplemented with 1% nonessential amino acids and 10% fetal bovine serum. The murine microglial BV-2 cell line was purchased from Istituto Nazionale per la Ricerca sul Cancro (Genova, Italy) and cultured in RPMI 1640 (Thermo Scientific) supplemented with 10% foetal bovine serum and l -glutamine. Primary neuronal cultures were prepared from cerebral cortices of embryonic day 15 mice using a dissociation solution (Sumitomo Bakelite, Tokyo, Japan). The cells (5 × 105/cm2) were plated on polyethyleneimine-coated dishes and cultured for 7 days in neurobasal medium with 25 mM KCl, 2 mM glutamine and B27 supplement (Thermo Scientific). For Transwell cultures, APP-expressing SH-SY5Y cells (5 × 105/cm2) cultured on 24-well plate inserts (0.5 µm pore; Corning, NY) and BV-2 cells (1 × 106) placed below the inserts were treated for 24 h with 10 µM GlcCer or ceramides in Eagle’s minimum essential medium/Ham’s F-12 medium.
5
Isolation and Characterization of Primary Mouse Neurons, Microglia, and Astrocytes
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The protocols for animal experiments were approved by the Animal Experiment Committee of Nagoya University. Primary neuronal cultures were prepared from the cortices of C57BL/6 mice embryos at embryonic day 17 (E17) as described previously [27 (link)]. Briefly, cortical fragments were dissociated into single cells in dissociation solution (Sumitomo Bakelite, Akita, Japan), and resuspended in nerve culture medium (Sumitomo Bakelite). Neurons were seeded onto 12 mm polyethylenimine-coated glass coverslips (Asahi Techno Glass Corp., Chiba, Japan). The purity of the cultures was greater than 95%, as determined by NeuN-specific immunostaining [28 (link)].
Microglia were isolated from primary mixed glial cell cultures prepared from newborn C57BL/6 mice at day in vitro (DIV) 14 using the ‘shaking off’ method, which has been described previously [29 (link)]. The purity of the cultures was 97 to 100% as determined by immunostaining for the Fc receptor. Cultures were maintained in DMEM supplemented with 10% fetal calf serum, 5 μg/ml bovine insulin, and 0.2% glucose. Astrocytes were purified from primary mixed glial cultures by three or four repetitions of trypsinization and replating. The purity of astrocytes was greater than 95%, as determined by GFAP-specific immunostaining [30 (link)].
Microglia were isolated from primary mixed glial cell cultures prepared from newborn C57BL/6 mice at day in vitro (DIV) 14 using the ‘shaking off’ method, which has been described previously [29 (link)]. The purity of the cultures was 97 to 100% as determined by immunostaining for the Fc receptor. Cultures were maintained in DMEM supplemented with 10% fetal calf serum, 5 μg/ml bovine insulin, and 0.2% glucose. Astrocytes were purified from primary mixed glial cultures by three or four repetitions of trypsinization and replating. The purity of astrocytes was greater than 95%, as determined by GFAP-specific immunostaining [30 (link)].
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