Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Abstract

Citation: Pan, Z., Zhao, X., Brotto, M. Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber. J. Vis. Exp. (60), e3415, doi:10.3791/3415 (2012 . However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear [5][6][7] .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 5,[8][9][10] . 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 9. 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 9,11,12 .

Elevated Orai1 expression mediates tumor-promoting intracellular Ca2+ oscillations in human esophageal squamous cell carcinoma

“…Time-dependent quenching of intracellular Fura-2 fluorescence in response to entry of Mn 2+ via the SOCE mechanism represents a quantitative measure for Orai1 channel activity: the steeper the decline in slope, the greater the channel activity[24, 25]. Compared to that in HET-1A cells (Fig.…”

“…Cultured cells were loaded with 5μM Fura-2 acetoxymethyl ester (Invitrogen, OR) for 45 min at 37 ° C following a previously published procedure [25]. [Ca 2+ ] i was monitored by fluorescence microscopy with a 40x objective (Nikon TE200 Super Fluo, N.A.…”

“…SOCE activity is presented as ΔF 350 /F 385 , the difference between basal and maximal values of F 350 /F 385 after addition of 2mM CaCl 2 in BSS solution. Alternatively, SOCE was measured through use of Mn 2+ quenching assay[25], which is based on measurement of the fluorescence of fura-2 at a Ca 2+ concentration-insensitive isosbestic point (F 360nm ). Briefly, KYSE-150 or HET-1A cells (1x10 6 ) were placed in quartz cuvettes and pretreated with thapsigargin (5μM) in BSS to deplete ER Ca 2+ stores.…”

Extracellular Membrane Vesicles Derived from 143B Osteosarcoma Cells Contain Pro-Osteoclastogenic Cargo: A Novel Communication Mechanism in Osteosarcoma Bone Microenvironment

“…Briefly, 143B cells were loaded with 1 μM Fura-2 AM, a fluorescent dye which binds to free intracellular calcium by incubating at 37 ο C for 30 minutes according to previously described methods [31]. The ratio of Fura-2 excitations at 340 to 350 nm and 375 to 380 nm of light corresponds to the intracellular calcium concentration [Ca ++ ].…”

Mitsugumin 53 regulates extracellular Ca2+ entry and intracellular Ca2+ release via Orai1 and RyR1 in skeletal muscle

“…Our lab previously established a method for the quantification of SOCE in skeletal muscle cells, using Mn 2+ -quenching of intracellular fura-2 fluorescence333435. Mn 2+ is known to permeate cells via a SOCE mechanism but is insensitive to both the surface membrane extrusion processes and SR uptake by Ca 2+ pumps.…”