Many students have encountered this problem. The generations of their own cells have been quite good for generations. However, after several generations, they found that their cells are not working, and the state is getting worse. For this problem, you can say with great certainty that your digestion has gone wrong. Every time you digest, the cells are greatly damaged. So, the longer the worse. It can be said that for cells that need to be digested, each digestion is the most crucial test for the survival or not of this cell.
In the process of digestion, after you add trypsin, all the cells are digested by trypsin, but we ignore the problem that the growth state of the cells is different, and the adhesion of each cell is different. Some stick to the wall is firm, and some sticks are not so strong. Therefore, letting all the cells digest the same time is not fair to the cells. They will be treated differently and of course have temper. . . Ha ha. Everyone strives for teaching in accordance with their aptitude. For example, try the "four-step digestion method" below. (taking indigestible cells as an example)
Specific operation
1). First, without adding trypsin, pour off the old medium and add a small amount of new medium to wash 1-2 times (the purpose is to wash away floating dead cells and the like as much as possible).
2). Then add a small amount of new medium and blow it directly. This time, the cells that are not firmly attached are blown down. After washing again with the medium, the resulting suspension was mixed and introduced into a new bottle. (You can introduce these into a new bottle to compare with the cells that have been trypsinized to see who is better to know the sensitivity of the cells to trypsin.)
3). Clean the remaining liquid in the bottle, add about 0.3ml of trypsin to wash it again, aspirate it, and add about 1ml of trypsin to digest. Digestion is observed under the microscope. When the gap between the cells and the cells is obvious, immediately absorb the pancreatin and clean the warhead. Add new medium and start to blow. Blow 2-3 times and then suck the suspension into the test tube or new. The bottle is temporarily stored. Wash with a new medium of about 2 ml and mix with the front. (You can also introduce new bottle cultures to compare with the back and front.)
4). Then add the trypsin just sucked in the bottle to the bottle and continue to digest the remaining adherent cells. Of course, you can also use new pancreatic enzymes, if you are richer, hehe. Continue to observe under the microscope, until the remaining cell gap is obvious, when the cells are independent, absorb the trypsin and add new medium to blow. The suspension is introduced into a new bottle culture (according to the actual situation).
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