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Fluorescence-based measurement of store-operated calcium entry in live cells: from cultured cancer cell to skeletal muscle fiber.

Pan Z, Zhao X, Brotto M - J Vis Exp (2012)

Bottom Line: As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn(2+) into the cells represents a measurement of activity of SOCE.The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells.The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca(2+) indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Confocal Microscopy and Cell Imaging Core, Robert Wood Johnson Medical School. panzu@umdnj.edu

ABSTRACT
Store operated Ca(2+) entry (SOCE), earlier termed capacitative Ca(2+) entry, is a tightly regulated mechanism for influx of extracellular Ca(2+) into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca(2+) stores. Since Ca(2+) is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear. The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca(2+) measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca(2+) using the ratio of F(340nm;) and F(380nm;) (510 nm for emission wavelength) of the ratiometric Ca(2+) indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca(2+) release and Ca(2+) extrusion, a Mn(2+) quenching assay is frequently used. Mn(2+) is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca(2+) pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn(2+) into the cells represents a measurement of activity of SOCE. Ratiometric measurement and the Mn(+2) quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca(2+) indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules.

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Fluorescence-based measurement of store-operated calcium entry in live cells: from cultured cancer cell to skeletal muscle fiber.

Pan Z, Zhao X, Brotto M - J Vis Exp (2012)

© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376931&req=5

Bottom Line: As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn(2+) into the cells represents a measurement of activity of SOCE.The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells.The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca(2+) indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Confocal Microscopy and Cell Imaging Core, Robert Wood Johnson Medical School. panzu@umdnj.edu

ABSTRACT
Store operated Ca(2+) entry (SOCE), earlier termed capacitative Ca(2+) entry, is a tightly regulated mechanism for influx of extracellular Ca(2+) into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca(2+) stores. Since Ca(2+) is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear. The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca(2+) measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca(2+) using the ratio of F(340nm;) and F(380nm;) (510 nm for emission wavelength) of the ratiometric Ca(2+) indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca(2+) release and Ca(2+) extrusion, a Mn(2+) quenching assay is frequently used. Mn(2+) is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca(2+) pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn(2+) into the cells represents a measurement of activity of SOCE. Ratiometric measurement and the Mn(+2) quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca(2+) indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules.

Show MeSH