I am working on L929 cell line. After revival of cell line, the flask was not fully confluent but cells are adhered just 40-50% for 3 days, so I trypsined the cells and gave it for spin. After that because of very less cells I couldn't see the pellet. So I took the suspension and transferred that to new flask (T75) and I added 8 ml of fresh media. Does the left over trypsin will effect the growth of cells? Do cells get adhere? Please help
When adding the trypsin to the cell must be within 2 min only after that adding media or albumin serum to stop enzyme effect.
I mean some prefer adding serum to enzyme stop effect and other prefer media.
so, when you jest add anyone to trypsin causes change ph that led to enzyme stop
After cell detaching, trypsin must be neutralized by adding fx. complete culture media (with serum) in volume min. 2x vol of trypsin, it is very important. Cells shuold be centrifuge to remove supernatant and resuspend in fresh media. Trypsin in culture medium may effect cells condition, but L929 cells are'nt very sensitive, so just replace medium next day, they could survive.
No worry. FBS contains trypsin inhibitors such as α1-antitrypsin. The question is the amount of FBS to "quench" trypsin. I use usually at least 2 volumes of 10% FBS to 1 volume of 0.25% trypsin. By the way, when working in serum-free medium, you can use trypsin inhibitor, for example, soybean trypsin inhibitor.
we are washing by FBS solution when transferring the detachment cell from surface bottle
and cell inside bottle also. But we can stop (trypsin)enzyme effect by add serum or media that change ph. Ph chance is reflected on enzyme activity(inhibition)
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