In the cell laboratory, all kinds of cell experiments will be conducted. If adequate preparations and precautions are not taken, it will lead to serious consequences. This article will mainly introduce the basic operations of cell experiments.
I. Cell recovery:
Non-primary cultured cells are generally cryopreserved in liquid nitrogen. When they need to be cultured, they should first be taken out of the liquid nitrogen to be revived. The principle of cell resuscitation - rapid melting: Cells frozen in liquid nitrogen at -196℃ must be rapidly melted to 37℃ to ensure that the ice crystals stored outside the cells melt quickly. This prevents the ice crystals from slowly melting and entering the cells to form recrystallization, which could cause damage to the cells.
The specific operation is as follows:
1. Preparations before the experiment
Preheat the water bath pot to 37℃ and place the culture medium containing 10%FBS in it for preheating. Wipe the surface of the clean bench that has been exposed to ultraviolet light for 30 minutes with 75% alcohol. In the ultra-clean workbench, arrange the sterilized centrifuge tubes, pipettes, culture flasks, etc. in order.
2. Remove the cryotubes and defrost them quickly:
According to the cell cryopreservation records, find the required cell numbers by label. Remove the cell box from the liquid nitrogen tank, take out the required cells, and check the numbers outside the tubes at the same time. Quickly immerse the cryotube into the preheated water bath to thaw it rapidly, and keep shaking it constantly to make the liquid in the tube melt quickly. After about 1-2 minutes, the liquid in the cryotube will be completely dissolved. Take it out and wipe the outer wall of the cryotube with an alcohol cotton ball, then put it into the laminar flow hood.
3. Balance centrifugation: Aspirate the cell suspension into a centrifuge tube and centrifuge at 1000 to 1500rpm for 3 minutes.
4. Prepare the cell suspension. Aspirate the supernatant, add 10 ml of culture medium, and gently pipette evenly to suspend the cells.
5. Cell counting: The cell concentration is preferably 5×105/ml.
6. Culture cells: Aspirate the cell suspension that meets the cell counting requirements into the culture flask, and place the culture flask in an incubator at 37 ° C and 5% CO2 for culture (slightly loosen the cap of the culture flask). The time for changing the medium depends on the condition of the cells. Generally, except for a few cells specifically noted as sensitive to DMSO, the vast majority of cell lines (including suspension cells) can be directly placed in a culture flask containing 10-15 mL (5-10 times) of fresh medium after thawing. The next day, the fresh medium can be replaced to remove DMSO. This can avoid the problem that most cells cannot grow or adhere after thawing.
Ii. Cell Passage:
When the cultured cells grow to a certain density, due to the gradual consumption of nutrients in the culture medium and the gradual accumulation of metabolites (it can be seen that the culture medium turns yellow), and the growth space of the cells is also restricted, it will affect the continued survival of the cells. At this point, it is necessary to separate out some cells and update the culture medium. This process is called subculture.
Passage of adherent cells
The specific operation is as follows:
1.1 Preparations before passage:
Preheat the culture medium: Place the bottle containing the culture medium, PBS and trypsin in a 37℃ water bath to preheat. Wipe your hands with 75% alcohol and the ultraviolet-exposed laminar flow hood. Properly place the equipment in use: Ensure sufficient operating space, which not only facilitates operation but also reduces contamination. Light the alcohol lamp: Be careful that the flame should not be too small. Prepare the sterilized empty culture flasks that will be used. Take out the preheated culture medium, wipe it with an alcohol cotton ball, and then place it in the laminar flow hood. Take the cells out of the incubator. When removing the cells, make sure to tighten the bottle cap. Wipe the surface of the microscope with an alcohol cotton ball, then observe the cells under the microscope and make records. The cell culture flasks should be wiped with 75% alcohol and then placed on the laminar flow hood.
1.2 Trypsin digestion:
After disinfecting the bottle cap over the flame of an alcohol lamp, open the cap, bring it close to the alcohol lamp, carefully draw out the old culture medium, wash it 2-3 times with PBS, and slowly add an appropriate amount of digestive fluid (trypsin solution) along the wall (the side without cells), gently shaking. Pay attention to the amount of digestive fluid to cover the cells. The optimal digestion temperature is 37℃. Observation of cells under a microscope: When observing digested cells under an inverted microscope, if the cytoplasm retracts and the cells no longer connect into patches, it indicates that the cells are being digested moderately at this time.
1.3 Blow and disperse cells:
Discard most of the digestive fluid (leaving only a small amount in the bottle), gently tap the culture flask to disperse the cells, and then add fresh culture medium.
Then gently pipette it into a cell suspension with a pipette (try not to produce foam, otherwise it will cause damage to the cells)
1.4. Aliquot and dilute the cells
Aspirate the cell suspension and aliquot it into 2 to 3 culture flasks. Add an appropriate amount of culture medium and tighten the bottle caps. Place the culture medium under a microscope to observe the cell count and count if necessary. Note that too low a density will affect the growth of passage cells. The density of passage cells should not be lower than 5×105/ml. Finally, make marks on the bottle, such as cell generation, date and name, etc.
1.5. Continuing training:
Wipe the culture flask with an alcohol cotton ball, loosen the cap appropriately, and place it in the CO2 incubator for continued culture. The passaged cells began to adhere 2 hours later
On the bottle wall. When the spread area of the growth cells accounts for 25% of the bottom area of the culture flask, it is a +; when it accounts for 50%, it is a ++; when it accounts for 75%, it is a +++.
2. Passage of suspension cells
Suspension cells can be directly aspirated from the culture flask, leaving only a small amount, and then fresh culture medium can be added. When there are many fragments or particles in the cell suspension and it feels rather dirty, the suspension can be transferred to a centrifuge tube and centrifuged at 1000 to 1500rpm for 3 minutes. Discard the supernatant, add about 2ml of culture medium, pipette until uniform, and then add 2 to 3 drops to a culture flask that has already been filled with fresh culture medium. Then place it in a carbon dioxide incubator for cultivation (loosen the cap of the culture bottle).
