From: Nebe-Von-Caron, G (g.nebe-von-caron@unipath.com)
Date: Thu May 01 2003 - 11:26:14 EST
That would go very much against the state of my office as a bounded region of space. Were I am there is increased entropy (e.g. chaos), but I can't be everywhere. Alternatively logic is: who works makes mistakes, who works a lot makes more mistakes; we make no mistakes As you might see from my earlier comment when it goes across neither the Principle Investigator (PI) nor the Food and Drug Administration (FDA) give you a guarantee for life. Indeed the presence of membrane integrity tested by propidium Iodide dye exclusion and the presence of metabolic activity (preferably an energy dependent measurement) gives you some degree of confidence that the cell still does something. Just be aware that the absence of the (carboxy)fluorescein retention can indicate a very efficient dye extrusion pump, in itself a good indicator for an energy dependent process. Biosynthesis is probably the most stringent parameter for functionality in the absence of reproductive growth, but in most cases we are just interested in excluding the permeabilised cells that interfere with our assays. However it seems obvious to me that the most active cells are the most likely ones to be killed by our preparation methods, so we should give that serious considerations. So always look at the bright side of life .. .. ........ Gerhard -----Original Message----- From: Mario Roederer [mailto:roederer@drmr.com] Sent: 29 April 2003 19:45 To: cyto-inbox Subject: "What is life?" ... you should always know! At 4:06 PM -0400 4/28/03, Howard Shapiro wrote: The only conclusion that can be drawn from this is that we should abandon the unqualified use of the term "viability" in cell biology in general and cytometry in particular, and describe the states of cells based on objective criteria such as capacity to form colonies, to catalyze various enzymatic reactions, to exclude or retain specified dyes, to maintain cytoplasmic and/or mitochondrial membrane potential, etc. -Howard 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." Based on this, probably the best fluorescence measurement of viability would be one that measures membrane potential (i.e., requires both integrity and active processes). Alternatively, a combination of PI (to ensure integrity) and FDA (to ensure enzymatic activity) would work, with the exception of those cells that actively take up PI (although, it should be easy to discriminate this process from membrane permeability simply by kinetics). 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. mr (PS, everyone can stop casting aspersions about my personal viability now!)
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