From: Keith Bahjat (kbahjat@ufl.edu)
Date: Thu Dec 10 1998 - 06:58:41 EST
In 1995, a paper was published in J Imm Methods (Douglas, R.S. et al:188(1995)219-228) where the authors stained cultured cells undergoing apoptosis with PI, then divided the sub G1 area into regions and sorted these events. Once sorted, these events (maybe cells, maybe not) had DNA extracted and run on an agarose gel which was then stained with Ethidium bromide, to demonstrate the laddering effect which had been demonstrated many times in apoptotic populations. Their finding was that the largest sub G1 population, which conaitned a majority of the sub G1 events, had NO DNA! Though this was not the main thrust of their paper (they were trying to provide a reproducible method for surface staining with DNA labeling), it is certainly relevant that many labs are measuring increases in debris (which apparently stains somewhat with PI) rather than fragmented nuclei. This is particularly disturbing considering that today, three and a half years after publication of this paper, I still read MANY publications that simply draw a region encompassing all sub G1 events and call these apoptotic cells. Most of this is debris, and last time I checked, no one had published that increased debris correlated with increased cell death (though it may!). It seems that this may be valid if you have a strong software modeling program to eliminate the debris (or use the technique in the paper of FL2-A vs. FL2-W, though my plots never look as clean as the ones they produced), but most papers just pull up a histogram in WinMDI and draw markers for Go/G1, S, G2/M, and sub G0/G1. Please show me why I am wrong in thinking these methods are invalid, or explain why these papers are accepted for publication. Thanks for anyone and everyone's insight. Keith Bahjat Lowly, feeble minded Graduate Assistant University of Florida College of Medicine Gainesville, Florida kbahjat@ufl.edu
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