Primary cell culture differs from cell lines in that the two methods of operation are different during the culture process. Here is a description of the six errors and corresponding methods that are prone to occur during the operation of primary cells.
1. Long-term water bath thawing
Since primary cells are very susceptible to the thawing process, they are swayed and dissolved in water when thawed in a 37 ° C water bath. After the cells are half-dissolved (do not wait until they are all dissolved), quickly take them to the biosafety cabinet or the clean bench. Use a 1ml gun, take out the prepared medium and put it into the cryotube, and then pump it into the flask. Repeat until completely dissolved
2. Dissolve the cells in the cryotube and centrifuge quickly. <br>If the primary cells are lysed immediately after centrifugation, the cells may be hit harder than DMSO. This method is not recommended. After dissolving in complete medium with serum, the flask was placed, and the next day, the solution was changed to remove DMSO.
3. Let the original cells grow up (100%) bottles (dish, plate)
When the primary cells are full, the cells will age. Since the primary cells are not 100% cell clusters, it is important to keep the mixed cells to a minimum. It is recommended that the cells grow to 90-95% for passage.
4. Passage with a large amount of trypsin
When the primary cells are passaged, they are digested with a low concentration of trypsin. Under the microscope, the cells begin to round and detach, and the trypsin is rapidly stopped. It is recommended to terminate with a complete medium containing 10% serum or a protease-derived protease terminator.
5. After cryopreservation after cell culture <br> After the primary cells are re-frozen, the cells will age and may also cause functional changes, so it is not recommended to freeze. Re-frozen cells are also the main cause of cell death and injury.
6. Primary cells are immortalized
Unlike primary cells and cell lines, proliferative capacity is limited. In order to prevent genetic changes, it is best to start experimenting as soon as possible. In addition, in order to prevent an increase in the proportion of mixed cells, it is necessary to observe the cell morphology frequently.
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