Of course I agree with Bill Telford's comments but would like to point out some of the benefits of DNA content measurements. Relative (or absolute) DNA content measurements using EtOH-fixed cells provide a wealth of information regarding cellular responses to any number of treatments that are not available in any other, single assay. The assay is also extremely inexpensive and convenient fix the cells and analyze after all the time points have been collected. The fact that it can also provide information regarding late-stage apoptosis (or other forms of programmed cell death) is an added plus. Quantifying sub-G1 (or sub-G2/M peak with apparent S-phase DNA content) at a given time point/treatment, is not always possible with any certainty, as Bill has articulated below. But then again, no other assay can quantitatively capture the kinetics of programmed cell death with certainty. DNA content can also reveal growth inhibition in the absence of programmed cell death by a decrease in S-phase cells and corresponding increase in G1 something that is completely missed if you are only looking for caspase activation or annexinV binding. To suggest that apoptosis assays other than DNA content are the panacea for enlightening our understanding of programmed cell death is delusional. TomD writing from home, currently at sanofi-aventis pharma/oncology > On Jun 8, 2006, at 3:47 PM, Telford, William ((NIH/NCI)) [E] wrote: > >> Just to add a couple of things to this good discussion... >> >> The appearance of the sub-G0/G1 is highly variable between cell types, >> and even the same cell type at differing levels of activation. Cycling >> cells in particular (with less compacted chromatin) are particularly >> likely to give confusing results, especially with the accumulation of >> subcellular objects (cell fragments, fragmented chromatin, other trash) >> at the low threshold of instrument sensitivity. The contribution of S + >> G2/M cell apoptosis to the "pot" further complicates the issue of data >> interpretation. And if we can't clearly separate intact apoptotic cells >> from apoptosis-associated debris, we are not really measuring >> cell-by-cell apoptosis anymore. >> >> We strongly encourage our investigators to use sub-G0/G1 ONLY as a >> preliminary, qualitative indicator of cell death, not as a quantitative >> assay - unless the apoptotic peak is really clear, which does not happen >> most of the time. It should always be backed up with additional assays, >> especially biochemical ones like caspase activation. And most journal >> reviewers, grant review panels, etc. won't be impressed anymore if DNA >> loss is the only criterion for measuring cell death - there are lots of >> other nice flow apoptosis assays available now, some of them fairly >> economical. >> >> Enjoy, >> >> Bill Telford >Received on Wed Jun 14 10:58:00 2006
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