Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

Cytometry
K H ElsteinR M Zucker

Abstract

Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we observed a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 microM tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 microM dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganizati...Continue Reading

References

Jan 1, 1992·Cytometry·Z DarzynkiewiczF Traganos
Mar 16, 1992·Biochemical and Biophysical Research Communications·T CromptonJ Tschopp
Aug 1, 1992·Biological Reviews of the Cambridge Philosophical Society·S Sen
Jan 1, 1986·Annual Review of Biophysics and Biophysical Chemistry·W G NelsonD S Coffey
Apr 8, 1986·Biochemistry·D Sen, D M Crothers
Mar 1, 1980·Analytical Biochemistry·C Labarca, K Paigen
Jul 1, 1994·Toxicology and Applied Pharmacology·R M ZuckerJ M Rogers
Nov 1, 1993·Journal of Cellular Physiology·J GongZ Darzynkiewicz
Jan 1, 1993·Cytometry·V N AfanasyevS R Umansky
Jan 1, 1993·Cellular Immunology·G MiglioratiC Riccardi
Mar 1, 1993·Immunology Today·J J Cohen
Apr 1, 1993·Alcoholism, Clinical and Experimental Research·S J Ewald, H Shao

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Citations

Sep 21, 2000·Neuroscience Research·S MizuhashiN Matsuki
Jul 1, 1997·The Journal of Steroid Biochemistry and Molecular Biology·J C SeegersB E Lehnert
Mar 29, 2001·Molecular and Cellular Neurosciences·U GalderisiA Giordano
Aug 31, 2000·Molecular Cell Biology Research Communications : MCBRC·E GiannoniG Ramponi
Jun 14, 2002·Experimental Cell Research·Hervé Lecoeur

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Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis