Primary cultures of cortical astrocytes can be applied to (1) cell preservation and (2) functional studies of cortical astrocytes.

Operation method

Attachment Discrepancy Method

Principle

Utilizing the characteristics of the existence of temporal differences in the growth of astrocytes, oligodendrocytes and microglia, different cell growth modes and cell adhesion to the culture layer, oligodendrocytes and microglia were removed from cultured mixed glial cells of cortical origin by using a constant temperature shaker at 37°C, with the strongest adhesion of astrocytes. The astrocytes obtained by this method were of high purity (more than 95%) and the cells had a good proliferative capacity.

Materials and Instruments

Neonatal 2-3d SD rat littermates
DMEM medium containing 10% fetal bovine serum D-PBS solution Digestive solution (0.25% trypsin + 0.04% EDTA)
37℃ constant temperature shaker

Move

1. Cultivate oligodendrocytes, wait for 10 days after the cell stratification, and the astrocytes have been completely spread to the bottom layer, start purification.

2 On a 37℃ constant temperature shaker, fix the culture flask and shake it at 280r/min for about 20h overnight.

3. On the next day, observe the shedding of the upper layer of cells under the microscope, if the upper layer of cells is basically removed cleanly, while the lower layer of cells are still intact and attached to the wall, the purification can be terminated. After discarding the culture medium, the cells were separated from the culture flask and inoculated again according to the method of cell passaging. Successfully isolated cells are free from contamination, in good condition, with fast cell proliferation, can be passaged in about 1 week, and without too many stray cells.

Caveat

1. When shaking overnight purification of cells, pay attention to seal the bottle mouth with sealing film to avoid contamination and air leakage.

2. If the cells in the lower layer start to de-wall in pieces after shaking overnight, the shaking should be terminated in time and the culture should be started.

3. The astrocytes re-inoculated after nitrification should preferably be inoculated on polylysine-treated medium, otherwise the cells will be easily clustered and grow in a bad condition.

Common Problems

1. When choosing animals for primary culture, it is best to choose neonatal 2-3d littermates, when the number of cortical astrocytes is the highest and easy to survive.

2. Primary culture of astrocytes can be passed on, but should ensure that the cells used in each experiment are of the same generation.

Source: Neurobiology Practical Experimental Techniques, Fourth Military Medical University Press.

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