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Determining the Extent of Toxicant-Induced Apoptosis Using Concurrent Phased Apoptosis Assays

Protocol
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

Apoptosis is a stage-dependent process exhibiting characteristic biochemical, molecular and morphological features that vary progressively through the apoptosis process. Apoptosis induced by toxicants may activate varied features of apoptosis to different extents and kinetics. Some of the features activated may occur transiently, while others may not occur in a cell system undergoing toxicant-induced apoptosis. Thus, the best approach for quantitating the extent of toxicant-induced apoptosis involves the utilization of a combination of assays focusing on different morphological, biochemical and molecular features of apoptosis. The kinetics of the apoptosis process will also need to be studied in any cell system subjected to toxicant-induced apoptosis for the first time, especially when more than one compound (such as an apoptosis inducing agent and an inhibitor) is being utilized. The use of multiple concurrent and phased apoptosis assays will streamline the process of determining the extent of apoptosis in such systems. Thus, in this report, we describe the concurrent use of an early, intermediate, and late apoptosis assay in order to measure different biochemical and morphological properties of apoptosis in the same system. We demonstrate the usefulness of the concurrent phased apoptosis assays using human TK6 lymphoblasts undergoing diepoxbutane-induced apoptosis. Additional notes, as well as tips for modifying the protocol for adherent cells, are included.

Key words

Apoptosis Apoptosis assay Concurrent phased apoptosis assays Lymphoblasts Caspase-Glo 3/7 assay pSIVA-IANBD apoptosis assay Nuclear morphology fluorescent dual dye staining assay 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Energy and Environmental SystemsNorth Carolina A&T State UniversityGreensboroUSA
  2. 2.Department of BiologyNorth Carolina A&T State UniversityGreensboroUSA

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