FACS analysis in adherent cell lines - FACS analysis in adherent cell lines (Jan/11/2005 )
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?
it depends on whether the protein can be digested with trypsin. If yes ,I don't think you can harvest the cells with trypsin.
Annexin-V binds to phosphatidyl serine (PS), which is a lipid that is flipped from the inside of the cell membrane to the outside during the early stages of apoptosis. Since PS is not a protein, you can easily use Annexin-V to visualize apoptotic adherent cells! Good luck!
You can use AnnexinV staining by FACs for adherent cells, but you gotta treat them very, very gently during the prep or you'll get loads of false positives due to mechanical damage. You don't want to trysinise the hell out of your cells, but it's worse to treat them roughly when detaching them - better to trypsinise for longer than to pipette too harshly. Ideally, you don't want to do either so this method may not be suitable for very adherent cell types, but you can tell from control samples - if the apoptotic index (AnnexinV-positive, PI-negative) goes above ~5% for your controls then you probably can't really take any reliable data from the experiment, though this will depend on the background levels of apoptosis of your particular cell line (e.g lowish for MCF-7 until they get anywhere near confluency, at which point lots of them will start floating). For something like HCT116 or LoVo you might find that you get up to 5% even under ideal culture conditions, so what would be relevant then is the increase in apoptotic index rather than the resting index.
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.