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2 protocols using b27 macs neuro brew21

1

Generating Mixed Glial Cells from Mouse Brains

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Mixed glial cells were generated from male and female P2-7 C57BL/6 pups according to the protocol of the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). In short, brains were dissected, cerebellum and meninges were removed and tissue was dissociated via mechanical and papain enzyme digestion prior to filtration through a 40 μm strainer. Cells were plated in poly-L-lysine-coated (10 μg/ml, Sigma) 96 well flat, glass-bottomed plates (BD Falcon) at a density of 105 cells per well and cultures were maintained at 37°C, 5% CO2. Cells were cultured for 5 days in DMEM (Life Technologies) supplemented with PDGFαα (10 ng/ml; PeproTech), 10% endotoxin-free FCS, 1% penicillin/streptomycin and 1% L-glutamine (Life Technologies). Cells were cultured for a further 2 days in neural medium (Miltenyi Biotec) supplemented with 1% penicillin/streptomycin, 1% L-glutamine, 2% B27/MACS Neuro Brew21 (Miltenyi Biotec) and PDGFαα (10 ng/ml; PeproTech). At day 7 in culture, PDGFαα was withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% Treg-conditioned media, rCCN3, anti-CCN3 (clone 231216) or isotype control (clone 54447) (all R&D Systems) or controls for up to 5 days.
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2

Generating Mixed Glial Cells from Mouse Brains

Check if the same lab product or an alternative is used in the 5 most similar protocols
Mixed glial cells were generated from male and female P2-7 C57BL/6 pups according to the protocol of the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). In short, brains were dissected, cerebellum and meninges were removed and tissue was dissociated via mechanical and papain enzyme digestion prior to filtration through a 40 μm strainer. Cells were plated in poly-L-lysine-coated (10 μg/ml, Sigma) 96 well flat, glass-bottomed plates (BD Falcon) at a density of 105 cells per well and cultures were maintained at 37°C, 5% CO2. Cells were cultured for 5 days in DMEM (Life Technologies) supplemented with PDGFαα (10 ng/ml; PeproTech), 10% endotoxin-free FCS, 1% penicillin/streptomycin and 1% L-glutamine (Life Technologies). Cells were cultured for a further 2 days in neural medium (Miltenyi Biotec) supplemented with 1% penicillin/streptomycin, 1% L-glutamine, 2% B27/MACS Neuro Brew21 (Miltenyi Biotec) and PDGFαα (10 ng/ml; PeproTech). At day 7 in culture, PDGFαα was withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% Treg-conditioned media, rCCN3, anti-CCN3 (clone 231216) or isotype control (clone 54447) (all R&D Systems) or controls for up to 5 days.
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