A photomicrograph of cultured embryonic rat hippocampal neurons after 10 days in vitro. The cells were loaded with 3 uM of Asante Calcium Red AM for 30 minutes in culture media then washed in dye-free media for 30 minutes.
Cells were perfused in a flow through chamber and imaged on the stage of an inverted Nikon microscope with a 40 x 1.35 NA objective. Cells were illuminated with Xenon bulb using a 35 nm wide band of light centered around 572 nm and a 60 nm wide band of light centered around 632 nm was imaged with a cooled CCD camera. The scale bar is 20 um.
The below graph shows the percent change in Asante Red Calcium fluorescence in the cell bodies of selected neurons in response to stimulation with NMDA (300 uM) in glycine-containing, Mg-free saline. Images were obtained every 20 seconds. Regions of interest were drawn around the somata, and mean intensities within the regions recorded as a function of time. Percent change of fluorescence at each time was calculated with respect to an average of the first five images.
Images, Graph, and description provided courtesy of J Marks, University of Chicago.
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
ORDER ACR HERE
Asante Calcium Red™ offers an alternative to GFP-Certified™ that produces a truly RED emission