You knowProcedure for subculturingIs it?
Subculturing is done by detaching cells from their original flasks and transferring them to new flasks for culture. This process is also known as subculture cells. Through subculture, cells can be expanded to form cell lines, which facilitate cell cloning and preservation, and provide a large number of experimental materials for scientific research.
SpecificProcedure for subculturingAs follows:
1.Removal of the original nutrient solution: Pour or remove the old culture solution from the flask.
2.Digest cells: Add a small amount of mixed trypsin solution and EDTA solution to the flask to cover the bottom of the flask. Place the flask at the appropriate temperature (incubator at 37 or room temperature at 25) for digestion. Observe the changes in the cells, and stop digestion as soon as the cytosolic retraction and intercellular space increase are found.
3.Rinse the cells: Aspirate the digest, add a small amount of Hanks solution, gently turn the flask, rinse the remaining digest before adding the culture medium. If only trypsin is used for digestion, the trypsin solution can be aspirated and the serum-containing culture can be added directly to stop digestion.
4.Detach the cells: Use an elbow pipette to gently tap the cells on the wall of the flask, repeatedly tapping them to detach from the wall of the flask and form a cell suspension. Be gentle when patting to prevent damage to the cells.
5.Seeding cells: After counting with a slide, the cells are seeded into new flasks and placed in a CO2 incubator for curing.
6.Replace the culture medium: According to the cell growth status and experimental requirements, determine the time of changing the culture medium. In general, change the culture medium every 2-3 days.
7.Once the cells have covered the bottom of the vessel, they can be used for experiments, or they can be continued for inheritance and maintenance to expand the scale, or they can be replaced with culture medium.
The following should be noted when subculturing:
1.Control the cell digestion time: Too little time will make it difficult for cells to detach from the flask wall, and too long will cause cells to fall off and be damaged.
2.Control the concentration of the digestive solution: when the concentration is too high, the digestion time should be shortened;When the concentration is too low, the cell digestion time is relatively prolonged.
After the primary culture is successful, with the extension of the culture time and the ** of the cells, contact inhibition will occur between the cells, and the growth rate will slow down or even stagnate. In addition, nutrient deficiencies and metabolite accumulation can also be detrimental to cell growth or toxicity. Therefore, subculture is required, and when subculturing cells, it needs to be strictly followedProcedure for subculturingand precautions to get the ideal experimental results.