Establishing an accurate and reusable neuronal culture model in vitro is of self-evident importance for experimental research in neuroscience. Many research groups have explored neuronal culture in vitro and developed some alternative experimental methods. Summarizing the results of these studies, we found that the simplicity of the experimental method, the expandability of the progeny cells, and the accuracy of maintaining the biological characteristics of the progeny should be taken into account when choosing the neuronal culture method.
Operation method
Chicken embryo neuron culture hands-on experiment
Materials and Instruments
Phosphate Buffer Poly D Lysine Hydrobromide Hank's Balanced Salt Solution without Calcium and Magnesium Ions DMEM with Glutamine Fetal Bovine Serum (FCS or FBS) B~27 Serum Free Additive Move I. Materials Incubate the white hen eggs in a pre-ventilated (forc-draft), humidified egg incubator (e.g. Agroswede, Malmd, Sweden) at 37.8°C for 8 d (or for a defined period of time). Eggs should be incubated with the blunt end facing upwards and automatically turned over every 2 h. The eggs should be incubated with the blunt end facing upwards. Note that the use of animal tissues requires legal approval. 1. Prepare and sterilize a borate buffer (2.37 g borax and 1.55 g boric acid dissolved in 500 ml of redistilled water, pH 8.4); 2. dissolve poly D lysine in borate buffer at a concentration of 50ug/ml; the 3. Appropriate amount of poly D lysine solution is applied to the inner surface of petri dish or culture bottle; 4. Incubate at room temperature for Ih or overnight; 5. Wash thoroughly with sterilized redistilled (distilled) water for 2~3 times; 6. Dry the culture vessels and then irradiate them under UV light for IOmin (choose the irradiation time appropriately). The coated culture apparatus can be stored at 4℃ for 1 week, but it is better to use it immediately. 1. Scrub small metal or plastic trays with a sterilizing solution containing 70% EtOH or 70% isopropyl alcohol. 2. Remove the egg so that the flat blunt end is facing upwards, as the air end of the egg is located at the blunt end, so that it is easiest to observe and remove the embryo through the membrane at that end. 3. Wipe the egg with ethanol or sterilizing solution, with the flat blunt end facing up, and place it in the box. 4. When all incubated eggs are sterilized and dry, transfer them to the ultra-clean table. 5. Add IOml of cold dissection solution to each of the three 60 mm plastic petri dishes (adjust the amount of dissection solution according to the number of embryos collected). 6. Crack and remove the egg shell above the air cavity at the flat blunt end of the egg with the dorsal side of the forceps and peel off the inner membrane. 7. Hold the neck of the embryo with curved forceps and carefully remove the embryo, taking care not to remove the yolk sac or its membrane with the embryo. 8. Place the removed embryos in a petri dish containing dissection solution. After all the embryos are collected, transfer them one by one in sequence to another petri dish for dissection and separation. 9. The chick embryo was placed ventral side down and fixed with the previously mentioned fine forceps through the dorsal aspect of the larger midbrain. 10. Partition the telencephalon and clip it out with a pair of fine serrated curved forceps. 11. Using forceps, place the removed telencephalon into a third Petri dish containing fresh dissection fluid. 12. When all telencephalons have been removed intact and transferred to the third petri dish, all meninges and blood vessels are carefully removed with sharp forceps. 13. Add 2 ml of dissection solution to a 15 ml conical tube and carefully transfer the isolated telencephalon tissue to this conical tube using a Pasteur pipette. 1. Blow the isolated telencephalon repeatedly with a long, sterilized pasteurized pipette to prepare a single-cell suspension. This is one of the more critical steps in the experiment and requires repeated practice to master well. When blowing, the tip of the pipette is inserted into the bottom of the conical tube, and the fragmented tissue is slowly sucked into the tube and then blown out at the same speed, which often requires more than 100~200 times of blowing back and forth. Since air bubbles can cause cell fragmentation, try to avoid blowing air into the liquid during the blowing process. 2. Switch to a pipette with a diameter twice as small and continue to blow 10~20 times at a faster speed and higher pressure. 3. The operation requires attention: try to use thick-walled pipettes during the blowing process, which can provide moderate blowing force; be careful not to use notched or cracked pipettes; the number of times of blowing depends on whether or not enzyme digestion is used and previous experience; do not try to blow all the visible tissue blocks into single-cell suspensions because some tissue blocks are composed of cell membranes and non-neuronal cells, which are not easy to be blown apart. 4. Dilute the single-cell suspension with 2x the volume (4 ml) of DMEM+20%FCS and repopulate with divalent cations and nutrients. Allow to stand for 3 min to allow the unblown tissue to settle. 5. Using a pasteurized pipette, carefully transfer the supernatant to another I5mI conical culture tube and centrifuge at 200Xg for one minute. 6. Discard the supernatant and gently add 5 ml of DMEM+20% FCS. gently blow the sediment at the bottom to resuspend it. This step removes some of the dead cell debris and cytotoxic substances released by them. 7. Place a 40/xm cell sieve at the top of a 50-mi plastic conical culture tube for single-cell suspension filtration, and rinse the sieve by aspirating 15-45 ml of DMEM+20% FCS through a 15-ml plastic pipette to remove aggregated cells from the suspension. 8. Take 50u1 of cell suspension and mix gently with an equal volume of Tapan Blue solution. Perform a viable cell count using a hemocytometer (bright cells that are not stained are viable). The number of cells will be affected by the gestational age and the ratio of the number of embryos to the volume of medium. In general, a good cell preparation is one in which the number of dead cells shown by Tapan Blue staining is controlled to be around 10%. 1. The density of cell inoculation should be decided according to the requirements of the experiment. Usually, high density inoculation is good for cell survival, but it will also increase the number of non-neurons. Comparing the results in different sizes of culture dishes and culture flasks, we recommend using the unit of number/area rather than number/volume as the unit of measurement for cell inoculation density. We recommend an inoculation density of 500?3000 cells/mm2. 2. The volume of medium required should be determined by the size of the petri dish or culture flask selected. For 35 mm, 60 mm, 100 mm petri dishes, generally add 2 ml, 3 ml, 10 ml of medium respectively. For T_25, T-75, and T-160 culture flasks, add 5 ml, 15 ml, and 30 ml of culture medium accordingly. For 96-well plates, 50-100% of medium is added to each plate, while for 24-well plates, 0.5 ml of medium is added to each well, 1 ml to 12 wells, and 2 ml to 6 wells. 3. Use a pipette with sterilized tip (or multi-channel pipette) to add appropriate amount of cell suspension into the pre-coated culture vessel, and place it in a 37 "C incubator with 5% CO2 for incubation. 4. Replace the culture medium (DMEM+5%FCS) within 24 hours. 5. After 48 hours of incubation, the culture medium was changed with serum white B27-free neur 〇 basalmedium. 6. Usually change the medium every 2~3d (Monday, Wednesday, Friday). 7. In order to obtain serum-free culture conditions that fully utilize the synthetic medium, care should be taken to completely remove the serum-containing medium when changing the medium. The serum-containing medium can be completely removed by using an electric suction device connected to a Pasteur pipette. Care should be taken in this step to move quickly in order to avoid drying out of the cells. It should also be noted that the liquid should not be added directly to the cell surface to avoid damage to the cells. Observation of cultured cells with a high-quality phase contrast microscope is the most important and obvious way to determine the viability of cultured primary neurons. In general, viable cells are wall-applied on the second day of inoculation. However, it is still difficult to determine whether the surviving cells are neuronal cells at this stage. After 3-4d of incubation, the cell bodies begin to flatten and become translucent, and many protrusions begin to form and stretch out to a length that exceeds the diameter of even a few cell bodies. At this stage, experienced specialists are able to determine the composition of the cultured cells, but it is more difficult for the average person. Staining techniques for certain neuron-specific molecular markers are required to determine whether a culture of the desired primary neurons has been successfully established. Immunocytochemical labeling of specific antigens is commonly used, and the staining technique employed during neurotomy is not recommended because its results are often inconclusive. Neuron-specific antigenic markers that are often used include neuron-specific xylanase (NSE), microtubule-associated protein (MAP), or neuron-specific antigen clusters [such as enzymes produced during transformation processes such as glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH)]. Antibodies to some of the neuronal markers are now commercialized and readily available. However, neuron-specific antibodies alone are not enough, and antibodies against other cell types are also needed as negative controls, such as glial cell-specific marker - glial fibrillary acidic protein (GFAP), which has been widely used for glial cell labeling and identification. Currently, a double labeling method is commonly used to identify cells in culture, i.e., neuron-specific and glial-specific antibodies are used to label the same cultured cells in parallel. However, the unique properties of different types of neurons (e.g., containing neurotransmitter synthesis and receptors) should not be taken for granted, and different models should be used for different purposes and rigorously controlled and validated. For more product details, please visit Aladdin Scientific website.
High Efficiency Air Particulate Filter CO2 Incubator
