cell harvest by scraping versus trypsinization - (Jul/13/2006 )
Just wondering, why do different cell lines have different protocol for cell detachment?
Are some cells just adhere more strongly than the others, thus needing trypsin?
Yes, some macrophage line needs 10+ min trysin treatment to get them off. Scraping maybe a better alternative.
true, some cells come off easier than others. Some need 2-3 min. and some more of trypsin.
Just out of curiousity what are the benefits of scrapping vs using trypsin OR using trypsin vs scrapping?
depends on what you're doing with the cells later, too
we work with HNEK. we use trypsin for most applications (passaging, internalization assays, etc), but we use scraping on ice for either protein or nuclear extraction protocols.
The problem with scraping is sometimes cells won't survive scraping and cannot attach again.
i would like to know what is the exact time for trypsinaization, i mean how long to leave trypsin before adding fetal serum?
i heared it depends on the cell type ? is this right?
yes. some takes 1 min, Most take 3-5 min @ RT.
I leave mine at 37C for around 3-4 min. But for one of my cell lines I need to leave it in 37C for 7 min.
i just got to know that trypsinization is not good to study kinaes and phosporilation status.. someone told me rthat this can be activated.
I would go for scrapping on ice as trypsinization might change the expression profile