Measurement of fluoride-induced endoplasmic reticulum stress using gaussia luciferase

Ramaswamy Sharma, Masahiro Tsuchiya, Bakhos A. Tannous, John D. Bartlett

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

Abstract

Endoplasmic reticulum (ER) stress and its consequent activation of the unfolded protein response (UPR) signaling pathway have been implicated in several pathophysiologic disorders as well as in drug resistance to treatment of tumors. Several techniques have been devised that qualitatively and quantitatively demonstrate the presence of ER stress and the activation of the UPR; however, most of these methods cannot be used to measure ER stress in real time. Here we describe the use of cells stably transduced with a secreted reporter, Gaussia luciferase (Gluc), to measure fluoride-induced ER stress. Factors that affect ER homeostasis, such as high-dose fluoride, will cause decreased Gluc secretion that can be measured as a decrease in Gluc activity in the culture medium supernatant. Gluc catalyzes the oxidative decarboxylation of coelenterazine (CTZ) to coeleneteramide, resulting in blue bioluminescence (λ max 485 nm). Therefore, Gluc activity can be easily quantified by mixing a small aliquot of the medium supernatant with CTZ and measuring the resulting bioluminescence in a luminometer. Among the various reporters used so far, Gluc is regarded as the most sensitive indicator of ER stress. A second advantage for using Gluc is its ability to function in a wide pH range. This is especially useful for studying fluoride-mediated toxicity as fluoride-induced stress is enhanced under acidic conditions. Since Gluc can be measured in a noninvasive manner, it has been used in several in vitro and in vivo applications. In this chapter, we detail our methodology for using Gluc to monitor fluoride-induced ER stress.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages111-125
Number of pages15
DOIs
StatePublished - 2011
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume491
ISSN (Print)0076-6879

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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