Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press
Operation method
basic program
Principle
Using the principle of layered growth of mixed glial cell cultures and semi-suspension growth of microglial cells, the uppermost layer of microglial cells in cortex-derived mixed glial cell cultures was shaken down by shaking method to continue cultivation for the purpose of isolation and purification of microglial cells.
Materials and Instruments
Neonatal 1-3d littermates Move 1 Follow the method of culturing oligodendrocytes, and leave the cells to grow in layers after 10d. Under the microscope, the round, refractive microglia can be seen in the full field of view, attached to the top of the astrocyte layer that grows close to the wall, tightly connected together and spreads all over the culture flask, and even many round microglia can be seen drifting down in the culture medium, and purification can be started at this time. 2. Fix the culture bottle into 37℃ constant temperature shaker, 80-200r/min, shaking for about 2h. 3 Collect the microglia cells from the shaking, plant them into polylysine-coated culture dishes, add new DMEM medium containing 10% fetal bovine serum and continue to cultivate, and change the medium every 2-3d. 4. Microglia are more difficult to divide and proliferate further in vitro, but if according to the experimental needs of subculture, one method can be used to scrape off the cells directly with a cell scraper, and inoculate them into a new culture dish; another method is to use 0.25% trypsin + 0.04% EDTA digestion, and then carry out subculture. The microglia obtained by this method can have a purity of more than 90%-95%, which can be identified by immunocytochemical staining with microglia-specific markers such as F4/80, IBA1 and IB4. Under the phase contrast microscope, the morphology of microglia in the resting state was short rod-shaped or elongated spindle-shaped with two or more protrusions and strong refractive properties, but under the activation and phagocytosis state, the morphology of microglia could undergo a great change, and the cytosol could become larger, longer or more rounded (Figures I--5). Caveat 1. All steps should be aseptic. Otherwise the microglia may be contaminated or activated by microorganisms and toxins.2 Before shaking the cells, make sure that the bottom layer of astrocytes is spread all over the culture flask and tightly connected, otherwise the astrocyte layer will be shaken up during shaking. Common Problems 1. After the microglia are separated by shaking, new culture medium is added into the original culture flask to continue the culture, and a large number of microglia will appear again after a few days, so that microglia can be collected by shaking repeatedly or even more times. 2. Due to the small proportion of microglia, it is difficult to divide and proliferate in vitro, so its yield is not high. It has been reported in the literature that the nutrient deficiency method or the addition of monocyte colony-stimulating factor (M-CSF) can be used to increase the yield. The authors added 15%-30% conditioned medium of L929, a fibroblast cell line that secretes M-CSF, resulting in sufficient microglia. For more product details, please visit Aladdin Scientific website.
DMEM medium containing 10% fetal bovine serum D-PBS solution Digestive solution (0.25% trypsin + 0.04% EDTA)
37℃ constant temperature shaker