Measuring calcium signaling using genetically targetable fluorescent indicators.

Measuring calcium signaling using genetically targetable fluorescent indicators. Research Abstract Details 

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Measuring calcium signaling using genetically targetable fluorescent indicators. Abstract Text:

Amy E Palmer,Roger Y Tsien,Amy E Palmer,Roger Y Tsien,

Genetically encoded Ca2+ indicators allow researchers to quantitatively measure Ca2+ dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca2+ in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca2+-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca2+ concentrations. Additionally, we highlight some of the challenges in observing organellar Ca2+ and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca2+ indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take approximately 1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take approximately 3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take approximately 1 d depending on the extent of analysis.

Measuring calcium signaling using genetically targetable fluorescent indicators. Publishing Authors By Initials

AE Palmer,RY Tsien,AE Palmer,RY Tsien,

For similar diagnosis: diagnostic techniques and procedures: diagnostic imaging: microscopy: microscopy, fluorescence research abstracts see: diagnosis: diagnostic techniques and procedures: diagnostic imaging: microscopy: microscopy, fluorescence research

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Measuring calcium signaling using genetically targetable fluorescent indicators. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Nature protocols

VOLUME: 1

Page Numbers: 1057-65

Journal Abbreviation:

ISSN: 1750-2799

DAY: 3

MONTH: 12

YEAR: 2006

Measuring calcium signaling using genetically targetable fluorescent indicators. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 101284307

Measuring calcium signaling using genetically targetable fluorescent indicators. Keywords Mesh Terms:

KEYWORDS: Microscopy, Fluorescence

MESH TERMS: methods

Chemical & Substance for Abstract: Measuring calcium signaling using genetically targetable fluorescent indicators. Information

Substance Name: Calcium

Registry Number: 7440-70-2

Grant and Affiliation Information for Measuring calcium signaling using genetically targetable fluorescent indicators.

AFFILIATION: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA. amy.palmer@colorado.edu

Country: England

AGENCY: United States NINDS

GRANT: NS27177

ACRONYM: NS

MEDLINETA: Nat Protoc

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