Hello Bob (and everyone).... I was very intrigued by your letter - extremely early apoptosis-associated cell volume flux is a very interesting characteristic of cell death. While a number of groups have published indirect evidence that this phenomenon occurs, not much has been done yet to demonstrate it directly by flow cytometry or other optical biology methods (at least in the published literature). Since the traditional flow cytometer is actually a rather poor instrument for measuring cell size, it is questionable whether a conventional flow cytometer can even measure what is likely to be a very small change in cell volume. We've poked at this issue over the last year or so and have made some preliminary observations. Keep in mind that these data are for mouse thymoma cells induced to die either by serum deprivation or by drug treatment. Other cell types are likely (aye, almost certain) to behave differently. (1) If you use fluorogenic caspase substrates to label multiple caspases simultaneously (such as 8 and 3), the negative, single-positive and multiple-positive apoptotic subpopulations all have slightly differing scatter properties. If looked at sequentially (like 8-3-, 8+3- and 8+3+, for example), the cell size appears to increase slightly during proximal caspase activation and return to "normal" after distal caspase activation. This is of course all much later than the phase of cell death associated with mitochondrial membrane potential flux, when the cell volume changes described by you are timed to occur. However, they are still much earlier than DNA dye permeability and annexin V binding. And this is MUCH earlier than the gross changes in cell size associated with late cell death. (2) If you sort various regions of the "scatter-viable" cell fraction (which of course really isn't all viable, but that's another story) and look at caspase activation, early DNA strand breaks, and other "early" characteristics of cell death, the percentages of cells with these cell death characteristics are not uniform across the entire scatter cluster. This suggests that the cells are altering their size as they progress toward later stages of cell death. (3) And finally, if we look at cell death via changes in mitochondrial membrane potential, we also see small changes in both forward and side scatter in "normal" and potential-perturbed cells. Again, the cells increase in size slightly, then seem to return to their original size. Of course, other causes (like cell cycle arrest) might be causing these changes as well. Cells are likely to undergo ion flux and subsequent volume changes during any perturbing stimuli, so its hard to say whether these changes are really part of apoptotic signalling or not. And finally, can we really see these changes with forward/side scatter measurement on a stock flow cytometer? Its no coincidence that we're now beta-testing Rick Thomas's NPE analyzer in the lab right now! Coulter volume measurement may be the only way to see these changes. If anyone has any data on this subject, I'd be very interested in hearing about it. Take care, Bill Telford At 12:43 AM 6/13/2002 -0400, Zucker.Robert@EPAMAIL.EPA.GOV wrote: >At a recent lecture on apoptosis, the hypothesis was put fourth that >cell volume accompanied by ion changes were the very significant and >initial events during apoptosis in lymphoid cells. It was suggested that >these changes came prior to caspase changes, mitochondrial changes, >membrane potential changes and nuclear fragmentation. > >1. What is the current thinking regarding the sequential events in the >apoptosis process that can be detected by fluorescent-based assays like >flow cytometry? > >2. After the lecture, I asked the speaker the question regarding the >capability of a flow cytometer to detect early size changes using light >scatter. Does the flow cytometer have a sufficient sensitivity to >measure early volume changes by light scatter? What is the percentage >decrease in cellular volume that would be necessary to effectively make >the conclusion that cell volume size changes are the initial event in >the apoptosis pathway? The speaker did not think this was a relevant >question and felt it was only a technical question in which the operator >of the equipment could easily answer. In my opinion in order to prove >the hypothesis that cell volume is indeed an initial event in the >apoptosis pathway, one has to understand how effective the flow >cytometer will be in determining early size changes using the light >scatter parameters. Can small changes in light scatter be detected early >in the apoptosis process and thus be used as an initial measurable flow >cyotmetric event in the apoptosis pathway? >Bob > > >Robert M. Zucker, PhD >U.S. Environmental Protection Agency >Office of Research and Development >National Health and Environmental Effects Research Laboratory >Reproductive Toxicology Division, MD 72 >Research Triangle Park, North Carolina, 27711 >Tel: 919-541-1585; fax 919-541-4017 >e-mail: zucker.robert@epa.gov
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