From: Howard Shapiro (hms@shapirolab.com)
Date: Tue Apr 29 2003 - 18:41:51 EST
Mario wrote- >Howard and David Novo are right in pointing out that this discussion >highlights the inaccuracy of the term "viability." > >On a more philosophical note, this discussion made me recall various >discussions I had with my father (a physicist) on what constitutes >"life". There is no need to enter into a very long and protracted >discussion about the various definitions and how they fall short (really, >don't do it). > >However, he did come up with a definition to which I still adhere, a >definition that is both pragmatic and practical: "An organism is alive if >it actively reduces entropy within a bounded region of space." So far, so good. Molecular Probes hasn't advertised a fluorescent probe for entropy yet, but stay tuned. >On a practical side, though, please do not let these ruminations dissuade >anyone in your labs from assessing viability by some FACS-based method! I >want to stress that EVERYONE should be routinely measuring viability in >some way in their experiments! I can't tell you how many times I have >discovered that some new subset or co-expression or population turned out >simply to be dead cells that have a nasty habit of nonspecifically binding >antibodies. Choose the viability stain that works for you, but please, do >it more than once a month, do it in every experiment! For this, PI is >very convenient because you can usually use it without dedicating any >additional detectors--just throw it in; the PI is usually so bright that >the dead cells can be gated out and then you can measure something in the >"FL3" channel. For this purpose, we keep aliquots of 100 µg/ml PI in PBS >in the freezer, and simply add it (100x solution) to our cells during >their normal stain. I agree in principle with the sentiments expressed in the above paragraph. However, after conceding the point of my initial comment on the inappropriateness of the term "viability", Mario goes right back to recommending that everybody measure "viability". In the context of the paragraph, he is talking about measuring loss of membrane integrity, using PI (or 7-AAD, or a Molecular Probes SYTOX dye, etc. ), when doing immunofluorescence measurements on *unfixed* cells (a luxury for many of us since the early 1980's). There is no question that failing to exclude cells with damaged membranes from analysis under these conditions will confound results due to the nonspecific binding of antibodies by such cells. By all means, use a dye to indicate membrane integrity or the lack thereof, as Mario suggests, but stop calling it a "viability" measurement! -Howard
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