Primary culture experiments of cortical/hippocampal neurons

Summary

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

Operation method

basic program

Principle

Neurons precede glial cells in development, so fetal mice are usually chosen for intracerebral neuron culture. El7-l8d pregnant rats or El4-16d pregnant mice are usually taken for neuron culture. Neonatal 1d littermates can also be used for neuronal culture, but there are more heterocytes after successful culture, and further purification is sometimes required. Cell culture methods are similar for these two sites

Materials and Instruments

El7-18d pregnant rats or E14-16d pregnant mice Newborn Id littermates
DMEM medium with 10% fetal bovine serum Neuronal maintenance medium (Neurobasal+B27+Glutamine)
Culture flasks

Move

1. According to the result of cell counting before centrifugation, resuspend the cells with a small amount of neuron maintenance medium first, after sufficient resuspension, replenish the liquid to the appropriate volume, inoculate the cells on the medium such as culture plate and slide according to the density of (2-4) ^x104/cm2 and place them in the incubator at 37℃.

2. Be careful to move gently when touching the cells during the incubation process, and avoid taking the cells out of the incubator after inoculation of Id. The contamination can be observed according to the color and brightness of the culture medium (for contaminated cells, the culture medium is turbid and yellowish; normally it should be clear and transparent). 1/3 volume of the medium should be changed after 3d, and the purity can be identified by immunochemical staining using neuronal markers. From day 5 onwards, 1/2 volume of the culture medium is changed every 2d, and can be used for experiments according to the maturity of the neurons.

3. If the purity of neurons does not meet the requirements of experiments, we can add cytarabine (final concentration of 5µM), and after 2 days of action, 1/2 amount of fluid change, and gradually remove.

Successfully cultured neurons have no contamination, good refractive properties under phase contrast microscope, little or no cellular debris, long protrusions, clear medium, spaced cells, no clusters, uniform distribution, and not too many stray cells. Not so well cultured cells, many stray cells, neurons in fair or poor condition, more cellular debris

Caveat

1. Neuron culture is often 1/2 amount of fluid change, the culture fluid should be fully rewarmed in 37℃ water bath before fluid change, cold stimulation and full volume fluid change stimulation will make the cell viability decline.

2. The older the rat is, the more gentle and delicate the operation of neuron culture should be, the key lies in the digestion process and blowing process. The effects of digestion time and blowing times on cell viability are the greatest in primary culture. Under the premise of ensuring that enough single cell suspension can be obtained, the operation time and steps should be shortened appropriately.

3. There is no big difference between the culture of hippocampal and cortical neurons, the cortex is rich in cells and it is easy to collect cells, but there are more stray cells.

4. Using pregnant rats for neuron culture, the purity is higher than that of newborn rats, and the vigor is better. When culturing hippocampal neurons, the gestation period is insufficient, the hippocampus is not yet fully formed, it is more difficult to separate, but it is okay to take the cortex.

Common Problems

Experimental insights:

1. applying acitretin to neurons, the cell purity will be higher. 1/2 amount of liquid exchange to remove, although there will be a residue, but the effect of such a concentration on neuronal activity is basically negligible.

2. When encapsulating slides, it is customary to place 8 slides in a small dish to which 2 ml of PLL has been added, add them one by one, and after adding them, blow them carefully with a curved tube, cover the dish with a lid, and shake gently. Then put them into the incubator for overnight encapsulation. At the end of the incubation, rinse with sterile water for 2 times, and clip out one piece by one piece with a curved camera, put it on the sterilized gauze, and blow-dry it in the ultra-clean table.

3. To inoculate the cells on the slide, prepare a single cell suspension at a concentration of (2-4) ^X105/100µL, inoculate 100µL on the slide, let it rest for 20min, then move it to the incubator and replenish the solution to 400µL after 2-3h. Cells inoculated in this way can be evenly distributed on the slide.

4. In order to avoid the drying out of the culture solution after a long period of incubation, sterile water can be added to the gap between each well of the culture plate.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/