CHIP - Harvesting cells by trypsin - (Apr/13/2006 )

Hi,

I am just starting with CHiP and I stumbled on the first step. When I scraped cells in PBS with protease inhibitors I lost qute a lot of cells because they could not be harvested. They were floating on the surface of the vessel and I could not rince them down. That never happened to me before. Also they were in clumps (our cells are always in clumps because we scrape them with glass beads, only prior to transfection we trypsinize them to get an accurate number for the dishes ) so the counting was quite inaccurate. Did you hear of any other way for collecting the cells. Can they be trypsinized?
Please help,
Grooya

Cells can be harvested by trypsin digestion. The problem is that after fixing with formaldehyde the cells are somewhat resistant to trypsin. Therefore, digestion takes a long time.

I scrape cells in media with added protease inhibitors. The other thing you can do is harvest cells in IP/Lysis buffer with added inhibitors. You have to assume some loss of cells.

Hope that helps.