Im fairly new to cell culture. I have been working with THP-1 cells. Its a suspension culture but upon inducing differentiation they attach pretty strongly. I am running into trouble when I harvest cells for RNA isolation.
I seeded the cells with various induction medias at 300K/ml density and 3e6 cells total in T75 flasks. I have attempted to detach the cells by incubating in PBS with 5mM EDTA at 37°C as per literature sources, but the cells do not attach even after 30 minutes. I ended up scraping them with a cell scraper. My RNA yeild and quality were not very good(RNeasy kit and nanospec), is this a product of scraping the cells? I should be getting more, as well as better quality RNA. The same number of cells harvested from un-induced cultures(suspension) provide much more and higher quality RNA.
Any suggestions for a better way to lift the cells, or a different approach to induction that would lead to better/more RNA.
Any help is appreciated.
Edited by BryanC, 19 May 2010 - 11:40 AM.