FACS analysis in adherent cell lines
Posted 11 January 2005 - 07:15 AM
I have previously worked with suspended cells in detection of apoptosis by FACS (annexin V). I would like to perform the same assay with adherent cell lines (such as MCF-7). Do anybody know if I can suspend cells with tripsin before annexin binding? In which conditions?
Posted 13 January 2005 - 08:48 AM
Posted 27 January 2005 - 03:31 AM
Posted 31 January 2005 - 04:55 PM
Non-confluent MCF-7 should be okay, but you want to gear your prep around using a smallish amount of trypsin for a short period, and then collect the cells using a 1 ml Liquipipette (you know the thing, a plastic squeezy-bulb disposable 1 ml thing - or anything with a wide-bore core, not a P1000 tip on a Gilsson - that gives massive false positives, particularly if you have to pipette up&down several times to get the cells detached). I've never used non-adherent cells for this so I don't know if leaving them hanging about for ages once you've stained them is detrimental, but for adherent cells you have to work fast as the longer you leave them sitting to stain on ice, the more false positives - I'd recommend against trying to process and analyse large batches of samples at the same time.