Chapter 3 Assays for DNA Fragmentation, Endonucleases, and Intracellular pH and Ca2+ Associated with Apoptosis

https://doi.org/10.1016/S0091-679X(08)61923-8Get rights and content
Under a Creative Commons license
open archive

Publisher Summary

This chapter discusses assays for DNA fragmentation, endonucleases, and intracellular pH, and Ca2+associated with apoptosis. Internucleosomal DNA fragmentation is measured by electrophoresis in an agarose gel. Most methods involve some sort of DNA purification, possibly involving separation of low from high molecular weight DNA, with phenol extractions, protease and ribonuclease digestions, and ethanol precipitations. These manipulations are not only time consuming but also limit any quantitative assessment of the amount of DNA fragmentation. Intracellular ion concentrations are measured using ion-sensitive fluorescent dyes. The best dyes for this purpose are those that have a shift in their fluorescence spectrum on binding the appropriate ion. The pH and Ca2+ in individual cells can be measured by flow cytometry. However, flow cytometry requires the use of “emission ratio” dyes. The selection of emission ratio dyes is more limited—carboxy-seminaphthorhodafluor (SNARF)-1 for pH measurements and indo-1 for Ca2+ measurements. The use of these dyes has clearly resolved subpopulations of cells undergoing ion changes.

Cited by (0)