From: Rackham, Don (drackham@guavatechnologies.com)
Date: Sun May 04 2003 - 14:24:21 EST
Yep...that pretty much sums up my life -----Original Message----- From: Pizzo,Eugene [mailto:Pizzo@NSO1.UCHC.EDU] Sent: Thu 5/1/2003 5:45 AM To: Cytometry Mailing List Cc: Subject: RE: "What is life?" ... you should always know! Mario Roederer, Actually I believe the definition of life requires direction or purpose so that a proteinoid Which serves to reduce entropy is not life but a proteinoid-like particle that actively Moves in the direction of a chemical gradient is. Gene Pizzo Manager, FACS Facility UCONN Health Farmington, Ct. 06032 Website; http://flowcytometry.uchc.edu <http://flowcytometry.uchc.edu/> -----Original Message----- From: Mario Roederer [mailto:roederer@drmr.com] Sent: Tuesday, April 29, 2003 11:45 AM To: Cytometry Mailing List 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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