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Measurement of [ca(2+)] (I) in whole cell suspensions using fura-2.
MedLine Citation:
PMID:  23007578     Owner:  NLM     Status:  In-Data-Review    
Use of Fura-2 in whole cell suspensions to measure changes in intracellular Ca(2+) is probably one of the simplest, yet most widely used protocols described in this volume. Whole cell suspensions are loaded with Fura-2 and then placed into a cuvette-based fluorimetric system (measuring 510 nm emission at alternating 340/340 nm excitation). Cells can be stimulated with agonists and antagonists to enable temporal response profiling and concentration-response curves to be constructed. The protocol can be used for a wide range of cells including those transfected with Ca(2+)-signaling proteins, e.g., receptors and channels. Loading characteristics and the need for agents to retain loaded dye (e.g., probenecid) need to be determined empirically. Calibration of whole cell suspensions to convert the fluorescent signal into Ca(2+) is simply performed using Triton-X lysis (to determine R (max)) and EGTA chelation (to determine R (min)).
Anish Patel; Robert A Hirst; Charlotte Harrison; Kazuyoshi Hirota; David G Lambert
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  937     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2013  
Date Detail:
Created Date:  2012-09-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  37-47     Citation Subset:  IM    
Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care, and Pain Management, Leicester Royal Infirmary, University of Leicester, Leicester, UK.
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