Primary culture of oligodendrocytes

Oligodendrocytes are derived from rats aged 1-2 days. After decapitation, the rat brain was collected in Ham’s F-12 culture medium, and the meninges and blood vessels were removed. The cortex was homogenized by mechanical homogenization, and then the cell suspension was filtered through 230 μm and 104 μm nylon meshes in sequence. The cells were collected by centrifugation at 1000 rpm for 7 minutes, and the cells were resuspended in DMEM supplemented with 12.5% ​​fetal bovine serum. Finally, the cells were inoculated into a Plastic Petri Dish at a concentration of 2.0×105 cells/cm2, and the Sterile Petri Dish Plastic was maintained at 37°C in a humid environment with 5% CO2. The medium was changed after 3 days, and every two days thereafter. After 9-11 days, the cells were able to cover the culture dish. The progenitors of oligodendrocytes were grown on the astrocyte layer and separated by shaking at 260 rpm for 18 hours on a rotary shaker, filtered through a 30μm nylon mesh, and then the cell suspension was inoculated into a culture dish containing a serum-free medium middle.
Medium composition: DMEM-Ham’s F-12 mixture (1:1), 10mM HEPES, 0.1% bovine serum albumin, 25μg/mL human iron transferrin, 30nM triiodothyronine, 20nM hydrocortisone, 20nM Progesterone, 10nM vitamin H, 5μg/mL insulin, 16μg/mL putrescine, 30nM selenium, 50U/mL penicillin and 50μg/mL streptomycin, and 2.5ng/mL platelet-derived growth factor AA and 2.5ng/mL Basic fibroblast growth factor is used to stimulate the proliferation of oligodendrocytes. The medium is changed every two days.
If in the absence of platelet-derived growth factor AA and basic fibroblast growth factor, primitive oligodendrocytes differentiate into oligodendrocytes in a serum-free medium, and 3% fetal bovine serum is added after 3 days. Both primitive cells and mature oligodendrocytes can be used to determine neurotoxicity.

https://www.huidalab.com/eo-sterlization-various-sizes-petri-dish-with-or-without-vents-product/


Post time: Dec-17-2021

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