Use of Annexin V in time-lapse microscopy / live imaging - (Aug/21/2006 )
I want to use Annexin to observe apoptosis in a live imaging experiment. This means that my cells are with a sitting in a microscope chamber for a few hours and I can't touch them. Importantly, I can't change the medium or wash the cells once the expriment has started.
My question is can Annexin V be used in such a system? Can I stain with Annexin V and not wash traces of it in the medium? Will the background level be OK?
How does Annexin V work anyway? I know it binds the membrane lipids, but does it "switch on" its fluoresence when it bind? Or do I see flu' just because of high conc' of Annexin at the membrane?
I dont think it will work for your system. FITC-annexin or other fluorescence labeled ones is typically used in this case, which means you have to wash the treated cells or the background will be too high.
It binds to anionic lipids which shows up on the outside of the plasma membrane when cells die.
it will be difficult since Annexin V need to be incubated in a "binding buffer"...
There is a paper from the lab of Douglas Green in which they did follow Annexin during live imaging (Lab Chip, 2005, 5, 628–633). Here is an extract form their methods:
"For microscopy experiments, the media was also supplemented
with 20 mM HEPES (pH 7.3) for pH buffering, 55 mM
2-mercaptoethanol for minimization of phototoxicity, and
2.5 mM CaCl2 for facilitation of Annexin-V binding. Dyes
(all from Molecular Probes) were added at the following
concentrations: tetramethylrhodamine ethyl ester (TMRE)
20 nM, YO-PRO-1 500 nM, Annexin V Alexa Fluor1 647
conjugate 0.5% (v/v)."
Could I be missing out on something? Is there some "trick" they have used to follow the cells?
Are you doing confocal? Confocol allows you to focus on a section of cell for which background may not be a serious problem (?!, not 100% sure). I would think this will be a real problem for conventional fluoresence microscope, unless you add low concentration of annexin to your samples.
I do not intend to use confocal microscopy. But it is not clear why this should make any difference. The main differences are that Confocal collects light only from a single focal plane, and that it uses lasers for excitation.
True, and the conventional microscope collects all the light in the path...
but the background is not limited to a sigle plane, so I will get background one way or the other.