Annexin, PI, 7AAD, viability, etc

Hello friends, 

I'm a bit late to the discussion, but better late than never. I've been doing viability studies for a living for some years now (if you can call my PhD's scholarship a living anyway...), and I've dug into these subjects quite a bit.

 

Regarding 7AAD, it was a great disappointment because the potential was really strong on paper. But it just doesn't turn out well enough (for me). I've tried and titrated it in a FACScan, FACScalibur and LSR II and the signal is always too low to be of practical use with other colors. I've done a triple stain of green fluorescence / TMRM / 7AAD and it was really tight, involving careful titrations and dye kinetic studies beforehand as well as very careful PMT settings. It was good enough for that specific experiment that we had to do but not versatile or flexible enough for day to day work, and we could resolve only the 7AAD positive vs negative and not the intermediate populations. When titrating 7AAD, at a given point the background just starts to climb without any increase in the positive vs negative separation. On the other side there are nice works out there showing its use, and when you look at it on it's own (without compensation and such), when properly titrated it's *almost* there. We seem to be about to buy a new cytometer for an eternity already, but if and when that time comes I definitely intend to make sure that we get a custom FL3 filter that is a bit broader than usual to obtain that extra fluorescence out of 7AAD. That is because 7AAD is otherwise a wonderful dye, with minimal compensation needs with usual FL2 filter configurations and almost no excitation by the red laser (violet does excite it more but the emission is outside anything but a Qdot filter set). You could do a great fluorescein, PE and 7AAD panel with only the 488 laser, which has the great advantage of using the most common (and cheapest) direct conjugates, minimizing compensation needs and leaving other lasers/detectors free, not to mention that it can be done on single laser machines.

 

On PMN and monocyte apoptosis using Annexin V, we have published a couple of months ago a paper dealing with exactly that subject, maybe some of our findings will be of use (you can find it here http://www.springerlink.com/content/u746703q6u57654n/) . The short story is that staining conditions can greatly affect your results, including medium used, calcium and phosphor concentration, time spent in incubation, and others. We have reason to believe the same is true for many (most?) other cells types. Different cells are affected to different extents, some being more or less "resistant" to these effects, and of course the treatments they have received affect as well their susceptibility. 

 

One thing I'd like to mention on Annexin V is that it is a high MW protein with a specific interaction site for a specific antigen. It sounds a lot like an antibody, and it should be titrated just like antibodies (or any other stain for that matter). In this case the titration's objective is not to reach saturation but rather optimal S/N. It can make a big difference (and provide nice savings as well).

 

Best regards,

 

Uriel.
Uriel Trahtemberg, M.Sc.
MD/PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL

"I wish to propose for the reader's favourable consideration a doctrine which may, I fear, appear wildly paradoxical and subversive. The doctrine in question is this: that it is undesirable to believe a proposition when there is no ground whatever for supposing it true."
 Bertrand Russell