Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2

Cell Calcium
Zuzana BerkovaM K Estes

Abstract

The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) ind...Continue Reading

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Citations

Oct 17, 2012·The Journal of Biological Chemistry·Shanshan SongQinghua Hu
Dec 15, 2010·MBio·Joseph M HyserMary K Estes
Jul 22, 2011·Virology Journal·Jose Luis ZambranoMichele E Hardy
May 22, 2009·Gastroenterology·Harry B Greenberg, Mary K Estes
Jan 29, 2009·The Veterinary Clinics of North America. Food Animal Practice·D M Foster, Geof W Smith
Jun 19, 2009·Cell Calcium·Yubin ZhouJenny J Yang
Aug 27, 2005·Journal of Medical Virology·Jesús Rodríguez-DíazJavier Buesa
Feb 18, 2011·Gut Microbes·Kim Hodges, Ravinder Gill
Jul 30, 2014·Current Opinion in Microbiology·Jinshu Fang, Pieter C Dorrestein
Oct 25, 2006·Biochimica Et Biophysica Acta·Mounia ChamiPatrizia Paterlini-Bréchot
Mar 10, 2016·Annual Review of Virology·Joseph M Hyser, Mary K Estes
Jul 28, 2016·PloS One·Sonja BialowasLennart Svensson
Aug 29, 2013·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Seung-Eon RohDavid E Kang

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