Posted 31 March 2010 - 02:48 PM
Hi, I read on the forums that over trypsinization will lead to cell lysis. People say that the cells break open, release DNA, and clump. But, I treated BJ fibroblasts with 0.25%Trypsin-EDTA for 30min at 37 degC and didn't really notice a difference in pellet size compared to BJ fibroblasts treated with Accutase for 10min at 37degC. I washed the cells a few times in PBS and counted them...the numbers were similar. The trypsinized sample did have a greater number of blue nuclei, but the cells themselves looked intact? So what exactly happens when you trypsinize cells for 30min? Does it pop open, permeabilize, or what? I'm trying to strip off proteins bound to the outside of the cell, so would acetic acid be a gentler method? What would be the most gentle yet most effective method? Thanks!