Asante Calcium Red Multiplexing with GFP and YFP
The following is an excerpt addressing the potential of ACR for multiplexing with Green fluorescent proteins or yellow fluorescent proteins
From a Poster Present at the 2010 SFN conferenece in San Diego, Ca.
Properties of Asante Calcium Red – a novel ratiometric indicator with long excitation wavelengths
Xenia A. Meshik*, Krzysztof L. Hyrc, Mark P. Goldberg**, Washington University School of Medicine, Dept. Neurology,
The Hope Center for Neurological Disorders, Alafi Neuroimaging Laboratory, Saint Louis, MO 63110
"...GFP and YFP, the Green and Yellow Fluorescent Proteins, are commonly used to identify cells successfully modified by genetic manipulations. The presence of YFP and/or GFP makes measurements of [Ca2+]i difficult as the robust protein fluorescence characterized by broad excitation and emission spectra, interferes with the signals of most commonly used indicators. Asante Calcium Red might be less prone to this problem as its emission wavelengths are much longer that those of GFP and YFP (D). To see whether this is the case, we compared changes in the indicator fluorescence in wild-type and GFP expressing neurons (A-C) during depolarization-induced [Ca2+]i elevation (F). The Ca2+- dependent indicator fluorescence (>560 nm) excited at 488 nm, a maximum GFP excitation, rose approximately 1.8 times in GFP-negative and 1.5 times in GFP expressing neurons (F). As the Ca2+-independent indicator emission (500-550) cannot be easily separated from the dominant GFP emission (D), it cannot be used as a reference in GFP/YFP expressing cells. Consequently, in the presence of GFP/YFP, Asante Calcium Red can work only as a single
wavelength indicator. Using different excitation wavelengths in the multi-photon excitation mode is not likely to separate the indicator and GFP fluorescence (E) but might help to isolate GFP-positive cells..."
View or Download the full poster HERE
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Asante Calcium Red™ offers an alternative to GFP-Certified™ that produces a truly RED emission