An easy ratiometric compensation for the extracellular Ca2+ indicator-caused fluorescence artifact.
Measurement of intracellular Ca(2+) dynamics is one of the most central real-time assays for cellular signaling. Ratiometric methods reduce the need for internal calibration and also effectively compensate for most artifacts when used in imaging. However, ratiometric calculation cannot compensate for extracellularly leaked (and fluorescent) Ca(2+) indicator and will instead indicate erroneous Ca(2+) concentration. This frequently occurs in systems where extracellular indicator is accumulated such as fluorescence spectrophotometers and plate readers. Here I present a method that, for the first time, fully compensates for this phenomenon. The method uses a single-step internal calibration together with a predefined ratiometric calibration protocol.
Kukkonen
Jyrki P
JP
Department of Basic Veterinary Sciences, Biochemistry, University of Helsinki, FIN-00014 Helsinki, Finland. jyrki.kukkonen@helsinki.fi
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling; D002138:Calibration; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D002118:Calcium