Asante Calcium Red loaded in embryonic rat hippocampal neurons
Posted on Tue, Jan 11, 2011
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.