Annexin-V-FITC or TUNEL?? - (Jun/07/2011 )
Hi Guys - hope you can help!!
I'm ordering a kit to use to detect apoptosis by FACS but I cant decide whether I should go for Annexin-V FITC or TUNEL?? Thing is my cells are quite clumpy - and whilst the clumpiness may cause a problem with Annexin-V - won't overzealous vortex/pipetting to separate the cells cause a major problem with TUNEL/both assays??
Help please asap!!
Clumpiness is going to cause a problem for acquiring in any channel (not just in your live/dead stain). Does filtration help with the clumping? There are these filters (like little nets) that you can place on top of your FACS tubes and you can pipette your cells into your tubes through the filters. I always thought it was really dangerous to be putting clumpy cells into the FACs machine. You could also use FSC-A vs FSC-H plots to rule out doublets/triplets, and only keep the singlets lying on the diagonal. From the singlets you could then gate for the live population. The most clumpy cells I've used were cells harvested from a liver explant that wasn't stored very well before it got into our hands. Hope this is helpful in some way =)