Primary culture experiments on cerebellar granule neurons
Primary culture experiments on cerebellar granule neurons
Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press
Operation method
basic program
Principle
The in vitro culture of cerebellar granule neurons is influenced by the site of sampling, special culture conditions and other factors, and it is easy to obtain cultures with high purity. The cells are bipolar protrusions and are commonly used to study the growth of neural protrusions. Its culture conditions have a high concentration of potassium ion-dependent properties and are often used as a research model for inducing neuronal apoptosis. When the culture medium of mature granule neurons is switched from high KCl (usually 25-30 mmol/L) to low KC! (the basic culture medium already contains 5 mmol/L KCl), the cells gradually undergo apoptosis.
Materials and Instruments
Neonatal 7d SD rat littermates Move Primary culture of cerebellar granule neurons from newborn 7d SD rats can be referred to the method of Moreno-Flores, the purity of cerebellar granule neurons cultured by this method reaches more than 95%. The specific operation procedure is as follows. 1. First, the cell suspension was obtained according to the general method, and then, in the cell precipitate obtained by centrifugation, the cells were resuspended by adding a small amount of complete culture medium (Neurobasal+B27+25mmol/L KCl), and then, according to the results of cell counting, the most appropriate complete culture medium was added. Cells were inoculated with polylysine-coated medium at the appropriate density for the culture purpose and incubated at 37℃ with 5% CO2. 2. On the 4th day of cell culture, 1/2 volume of the culture medium was changed, and the cells were basically mature on the 7th day. Cerebellar granule neuron cell body is small, about 24h after inoculation, began to extend the protrusion, the protrusion is easy to form a bundle growth. Figure I-2 shows the cell growth after 48h of inoculation. Caveat 1. In primary sampling, due to the sampling site and the size of the litter, the sterilized litter is usually placed in a 100mm glass dish, and the intact cerebellum is removed directly with dissecting instruments without severing the head. 2. The meninges of the cerebellum are not easy to be stripped clean, and should be removed especially carefully, otherwise it is difficult to remove the contamination of fibroblasts in the culture. 3. Due to the different purposes of culturing cells, there is a difference in the density of inoculation. If used for neural protrusion observation, can be inoculated (1-3)x105/cm2If used for the study of apoptosis induction, inoculate (4-6)x10 5 /cm 25/cm 22For apoptosis induction studies, inoculate at a density of (4-6) x 10 5 /cm 2 . 4. When doing apoptosis induction, it is usually enough to change the culture medium containing high potassium to ordinary Neurobasal+B27 culture medium without additional KCl. The number of apoptotic cells can reach about 40% after 8h according to the method of nuclear consolidation judgment. Common Problems 1 Cerebellar granule neuron cell body is small, the protrusion is long and thin, and easy to grow in bundles, I feel that the cell can do the general observation of the protrusion growth, and is not suitable for the use of software to analyze the length of the protrusion. The neurons can grow well in 3-4d, so we can start to observe. 2. After the cells have matured in vitro for about 7d, apoptosis experiments can be carried out. If you add a protective agent to inhibit apoptosis, you should be careful not to induce apoptosis for too long, otherwise it may lead to the failure of the protection experiment. For more product details, please visit Aladdin Scientific website.
10% fetal bovine serum+DMEM Neurobasal+B27 KCl
Culture Bottle