Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity

Anna K. Larsen, Arnaldur Hall, Henrik Lundsgart, S. Moein Moghimi

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Cationic polyplexes and lipoplexes are widely used as arti fi cial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully de fi ne the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fl uorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages23-33
Number of pages11
Volume948
DOIs
StatePublished - Dec 1 2013

Publication series

NameMethods in Molecular Biology
Volume948
ISSN (Print)10643745

Keywords

  • Apoptosis
  • Caspase activity
  • Cationic drug carriers
  • Cell-death assays
  • Gene therapy
  • Necrosis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

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