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Ratiometric Ca2+ Measurements Using the FlexStation® Scanning Fluorometer
Many commercial organizations currently use the Fluorometric Imaging Plate Reader (FLIPR®: Molecular Devices, Sunnyvale, CA) to conduct highthroughput measurements of intracellular Ca2+ concentration (see Chapter 7 ), taking advantage of its rapid kinetics, reliability, and compatibility for automation. For the majority of industrial applications, the primary limitation of FLIPR (i.e., its requirement for single wavelength fluorescent probes using visible light excitation) is not a significant issue. Indeed, visible light probes offer certain benefits over their ultraviolet (UV)-excited ratiometric counterparts, such as reduced sample autofluorescence and higher absorbance, thereby allowing relatively low concentrations of dye to be used. However, under certain circumstances researchers may prefer to conduct high-throughput experiments with ratiometric dyes, particularly when issues of dye leakage, photobleaching, or signal-to-noise ratio become a concern.
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Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3
The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.
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FlexStation examination of 5-HT3 receptor function using Ca2+- and membrane potential-sensitive dyes: Advantages and potential problems
The FlexStation is a 96 or 384 fluorescent plate reader with the capability of adding solutions during readings; it therefore has the potential to provide high throughput analyses of the functional characteristics of neurotransmitter-gated ion channels that can be examined using changes in fluorescence. The 5-HT3 receptor is one such protein, as its activation results in a change in membrane potential due to the opening of a Ca2+-permeant, cation-selective channel; it can therefore be studied using both Ca2+- and membrane potential-sensitive fluorescent dyes. Here we have used the FlexStation to examine the function of recombinant 5-HT3 receptors expressed in HEK293 cells using both these classes of dye. The results show that the pharmacological characteristics of the receptor obtained using the FlexStation is similar to those reported using other functional methods, although caution must be applied when using the membrane potential dye as large changes in membrane potential can yield inaccurate EC50s. Modifying the constituents of the buffer, however, so that the change in membrane potential was reduced, yielded EC50 values that were similar to previously reported data. We conclude that the FlexStation is a useful tool for high throughput studies when examining the function of neurotransmitter receptors that result in either a change in Ca2+ concentration or membrane potential.
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