Our operation methods, details, and theoretical explanations will become the guidelines for your operation, such as the placement of nutrient solutions and cell flasks; sterilization procedures; To learn their correct operation, it is necessary to pay attention to them the first time.
Nurturing cells like raising children
Cells are really fragile. It is best to check it every day to prevent abnormal phenomena such as lack of water, carbon dioxide, power failure, and insufficient temperature in the incubator. It is also necessary to solve these accidents in time to avoid greater pain caused by repeated experiments. .
Good cells should be seeded in time
Cells are passaged in batches so that even if one batch fails, there is a spare. It may not be easy for the average person like the latter. But this is the lesson of my blood. Once the cells were contaminated, the whole army was wiped out. At that time, I regret that I didn't protect the seed.
Each cell has its own characteristics and should be treated differently.
Cells are the same as humans, and different cells have different culture characteristics. Be careful in the cultivation process. Different cell lines use different media and serum.
For example, the smooth muscle cells I raise are very active and like to be lively. In 2-3 days, there will be a large population, and I don’t like to eat and drink very much. It is really the best child to raise. Endothelial cells and smooth muscle cells have similar personalities, but they are very unhygienic and sloppy. There are many secretory cells in them, so they need to be changed and washed frequently. The RAW264.7 cells are also very cheerful and very edible. It is necessary to feed them often. The headache is that the cells are easily activated. It is good that the bottle and environment in which they are cultured are free of endotoxins, but it is difficult for me as a mother to satisfy them.
Preparing for work before training
Including the handling of consumables, the configuration of reagents, sterility testing, etc.
Cleaning of glassware. For glassware (cell bottle, bottle containing nutrient solution, small penicillin bottle, etc.), first use washing powder to clean (pay attention to dead corners), and then rinse. Soak in acid solution for more than 24 hours, then rinse with clean water 10 times to remove residual acid solution. Bottles, etc., shake vigorously during rinsing. Then soaked in double distilled water for 24 h. After drying, wrap it up and dry it at 160°C for 2 hours.
Reagent configuration. As a liquid for cell culture, generally, more than double-distilled water may be used. And pay attention to the adjustment of pH value.
Sterility testing. Mainly use filter sterilization: such as trypsin, antibiotics, G-418 solution, various media, sodium pyruvate, glutamine, etc. Some can be autoclaved: PBS, D-Hank's, etc.
Reagents are stored in aliquots
Including nutrient solution, pancreatin, serum, etc.
Serum is especially important. Serum must be stored at -20°C. If one bottle cannot be used up at a time, 40-50ml can be dispensed into sterile acid-treated bottles, or even stored in penicillin bottles. Generally, the serum provided by the manufacturer is sterile and does not require sterile filtration. If a lot of suspended matter is found in the serum, the serum can be added to the medium and filtered together, and the serum should not be filtered directly. (No effect on cell culture under normal circumstances)
The bottled serum must be thawed gradually: 4°C refrigerator is completely dissolved and then divided into packaging. During the dissolution process, it must be shaken regularly (be careful not to cause air bubbles) to make the temperature and composition uniform and reduce the occurrence of precipitation. Do not thaw directly from –20°C to 37°C, as the temperature changes too much, which may easily cause protein coagulation and precipitation.
Heat inactivation refers to heating the fully thawed serum at 56°C for 30 minutes. After the water bath rises to 56°C, put the serum in, wait for the temperature to rise to 56°C and count the time, generally shake it evenly once every 5 minutes or so. The purpose of this heat treatment is to deactivate the complement components in the serum. Unless necessary, this heat treatment is generally not performed because it will cause a significant increase in sediment and affect the quality of the serum. Pay attention to the effect of changing the serum (including different batches) on the cells.
Strong sense of sterility
Before the experiment, the ultra-clean bench was sterilized by UV light irradiation for 30 minutes, the aseptic operation surface was wiped with 70% ethanol, and the ultra-clean bench fan was turned on for a few minutes before the experimental operation was started.
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