i trypsinized with 2ml and incubated for 5 minutes. only some cells are detached. what should i do next if this situation happened again after this?
Sure it is normal as long as it attached to the bottom.
FBS in media contains protease inhibitors, such as α1-antitrypsin and α2-macroglobulin. These inhibitors stop the trypsinization process and act by inhibiting lysosomal peptidases that may be released. Thus, it is essential to wash the incubated cell with PBS to remove FBS. Then only cell will detached.
It depends on what cells you are dealing with I guess. Some normal cells need more than 5 minutes to fully detached. But usually I keep them under 10 minutes. After 5 minutes, I will tap the flask gently and then leave it for 2 to 3 minutes in incubator and tap it again after that. But you do have to make sure you wash with PBS 3 times at least. And also as what has been mentioned by Sush, check your EDTA %.
oh. maybe it one of the reason. the cells are confluence and i just washed them twice. maybe i should wash 3 times with much volume of PBS. thanks everyone.
Can anybody provide me the reference for following statement as soon as possible please "FBS in media contains protease inhibitors, such as α1-antitrypsin and α2-macroglobulin. These inhibitors stop the trypsinization process and act by inhibiting lysosomal peptidases that may be released"
I am urgently looking for the reference to quote in my paper so any help in this regard is highly appreciated.
Many Thanks
rajesh kumar, maybe you should click on this link
1. http://www.labome.com/method/Cell-Culture-Media-A-Review.html#ref36
2. http://www.ncbi.nlm.nih.gov/pubmed/1368819
i'm not sure if it will help you or not.
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