Determination of in situ dissociation constant for Fura-2 and quantitation of background fluorescence in astrocyte cell line U373-MG

doi:10.1016/S0143-4160(97)90047-6

Cell Calcium

Volume 21, Issue 3, March 1997, Pages 233-240

https://doi.org/10.1016/S0143-4160(97)90047-6 Get rights and content

Abstract

Accurate estimates of cytosolic free Ca²⁺ with fluorescence indicators are dependent on the determination of the in situ dissociation constants (k_d) of the intracellular dyes and the correction for background fluorescence. The in situ dissociation constant for Ca²⁺ and indicator dye Fura-2/AM varies significantly from the in vitro published values due to differences in ionic strength, pH, viscosity and Ca²⁺ buffering by intracellular lipids and proteins. During the course of a measurement, background fluorescence changes may occur as the result of endogenous fluorescent compounds and compartmentalized Fura-2 indicator. The in situ dissociation constant value was determined for human astrocyte cell line U373-MG by creating several known intracellular Ca²⁺ concentrations while measuring total fluorescence and background fluorescence values for each. The background fluorescence was not constant, rather it demonstrated a linear relationship with the free cytosolic Ca²⁺ concentrations and total fluorescence intensities. The Ca²⁺ dependent and total fluorescence dependent background was expressed as a linear equation and subtracted appropriately from the total intensity measurements. The in situ dissociation constant was determined to be 3-fold greater than in vitro measurements after the background was corrected. The experimentally determined standard linear equations for background quantitation and the in situ dissociation constant for this line produce accurate cytosolic free Ca²⁺ estimates.

References (26)

G. Grynkiewicz et al.
A new generation of Ca²⁺ indicators with greatly improved fluorescence properties
J Biol Chem
(1985)
L.A. Blatter et al.
Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2
Biophys J
(1990)
L. Hove-Madsen et al.
Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca²⁺ binding properties
Biophys
(1992)
M. Konishi et al.
Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements
Biophys J
(1988)
J.W. Bassani et al.
Calibration of indo-1 and resting intracellular [Ca²⁺]_i in intact rabbit cardiac myocytes
Biophys J
(1995)
A.B. Harkins et al.
Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3
Biophys J
(1993)
N. Kurebayashi et al.
Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers
Biophys J
(1993)
D.M. Bers et al.
A practical guide to the preparation of Ca²⁺ buffers
Methods Cell Biol
(1994)
H. Ariyoshi et al.
Spatial distribution and temporal change in cytosolic pH and [Ca²⁺] in resting and activated single human platelets
Cell Calcium
(1995)
P.M. McDonough et al.
Measurement of cytoplasmic calcium concentration in cell suspensions: correction for extracellular Fura-2 through use of Mn²⁺ and probenecid
Cell Calcium
(1989)

J.P. Kao

Practical aspects of measuring [Ca²⁺] with fluorescent indicators

Methods Cell Biol

(1994)

T.R. Hesketh et al.

Free cytoplasmic calcium concentration and the mitogenic stimulation of lymphocytes

J Biol Chem

(1983)

J.P. Kao et al.

Photochemically generated cytosolic calcium pulses and their detection by fluo-3

J Biol Chem

(1989)

Cited by (37)

Exploring urinary bladder neural circuitry through calcium imaging
2023, Neuro-Urology Research: A Comprehensive Overview
With the advent of advanced microscopic imaging tools, measures of neuronal function are no longer limited to action potential traces on a polygraph. The introduction of calcium-sensing dyes and genetically encoded fluorescent calcium indicators now allow for real-time images of signal transduction within and between neurons throughout the body. In this chapter, we will explore the imaging tools used for measuring calcium signaling and the myriad of ways these tools can be implemented to image calcium signals in live neurons—particularly in the bladder and associated neural circuits. We will first provide an overview of traditional, nonimaging means of recording neuronal activity, as many of these techniques still have important uses today. Next, we will introduce the basics of calcium dynamics in neurons and identify the cellular processes driven by fluxes in calcium as opposed to sodium. We will then examine the implementation of calcium imaging dyes and genetically encoded calcium indicators in neuronal cell populations to show spatiotemporal patterns in excitability as shown by their calcium signals. We follow this with the pros and cons of how the most state-of-the-art imaging techniques are being used in the urinary bladder to examine not only single-cell excitability, but also entire sections of neuronal circuitry both ex vivo and in vivo. Finally, we will move beyond our current toolbox and explore promising new tools and techniques that potentially will allow never-before-seen visualization of membrane potential and ion flux throughout a neuron.
An efficient and rapid synthesis route to highly fluorescent copper microspheres for the selective and sensitive excitation wavelength-dependent dual-mode sensing of NADH
2021, Sensors and Actuators, B: Chemical
Citation Excerpt :
In many cases, this change is a shift in the absorption/excitation wavelengths [32]. Changes in fluorescence on specific ion/molecule binding can result in i) Increase/decrease in the quantum yield of the probe with little change in the excitation or emission spectra [33] ii) a shift in the absorption, and therefore the fluorescence excitation spectrum, to shorter/longer wavelengths with little shift in the emission maximum [34,35], and iii) a shift in both the absorption (excitation) and emission spectra to shorter wavelengths [36]. A common feature of fluorescent probes undergoing excitation shift is the presence of a unique “isosbestic point” [37] at which the fluorescence of the probe is independent of the ion/molecule binding.
Fluorescent copper microspheres (CMS) with high quantum yield (72.9 %) were synthesized by an efficient, one-pot, and rapid aggression-induced mechanism. The CMS was bright brick-red luminescent under UV light and have shown an excitation independent emission at 670 nm. The emission intensity of CMS was significantly quenched by the addition of nicotinamide adenine dinucleotide (NADH) when excited at 275 nm, providing a linear range with the logarithmic concentration of NADH, suitable for the detection of low concentrations of NADH. Similarly, the emission intensity at 670 nm was enhanced when excited at 340 nm, providing a linear range for NADH determination in the higher concentration. Thus, a dual-mode sensing platform for the determination of NADH was established by utilizing the fluorescent quenching and enhancement properties of NADH over CMS. Addition of other common interfering molecules and ions induced only a meager change in the fluorescent intensity of CMS, suggesting the high selectivity of the sensor. The linear range observed for NADH by exploring the fluorescent quenching properties was from 24.8−575.2 μM (R² = 0.99) with a L.O.D of 2.4 μM. Meanwhile, the linear range observed for NADH by utilizing the fluorescent enhancing properties was from 625 μM-2.30 mM (R² = 0.98) with a L.O.D of 322.5 μM.
The Oncogenic PRL Protein Causes Acid Addiction of Cells by Stimulating Lysosomal Exocytosis
2020, Developmental Cell
Extracellular pH is usually maintained around 7.4 in multicellular organisms, and cells are optimized to proliferate under this condition. Here, we find cells can adapt to a more acidic pH of 6.5 and become addicted to this acidic microenvironment by expressing phosphatase of regenerating liver (PRL), a driver of cancer malignancy. Genome-scale CRISPR-Cas9 knockout screening and subsequent analyses revealed that PRL promotes H⁺ extrusion and acid addiction by stimulating lysosomal exocytosis. Further experiments using cultured cells and Caenorhabditis elegans clarified the molecular link between PRL and lysosomal exocytosis across species, involving activation of lysosomal Ca²⁺ channel TRPML by ROS. Indeed, disruption of TRPML in cancer cells abolished PRL-stimulated lysosomal exocytosis, acid addiction, and metastasis. Thus, PRL is the molecular switch turning cells addicted to an acidic condition, which should benefit cancer cells to thrive in an acidic tumor microenvironment.
Transient Receptor Potential V Channels Are Essential for Glucose Sensing by Aldolase and AMPK
2019, Cell Metabolism
Fructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD⁺ levels in aged muscles, rejuvenating the animals’ running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.
In vitro electroporation detection methods – An overview
2018, Bioelectrochemistry
Citation Excerpt :
In the case of indo-1, the excitation is single (EX335) and the ratio between the fluorescence intensities at two emissions (EX405 and EM485) is determined [180]. Most measurements are done in a qualitative manner, although the concentration of free cytosolic Ca2 + can be determined quantitatively by in situ calibrations with solutions of known ion concentration (in combination with chelators) and ionophores for manipulating intracellular Ca2 + concentrations [181]. However, in experiments in which electroporation of a plasma membrane is determined by Ca2 + uptake, one must always be aware of the possibility that Ca2 + is also released from internal cellular stores, especially in the case of nsEP use [92,174,182].
Exposing cells to an electric field leads to electroporation of the cell membrane which has already been explored and used in a number of applications in medicine and food biotechnology (e.g. electrochemotherapy, gene electrotransfer, extraction of biomolecules). The extent of electroporation depends on several conditions, including pulse parameters, types of cells and tissues, surrounding media, temperature etc. Each application requires a specific level of electroporation, so it must be explored in advance by employing methods for detecting electroporation. Electroporation detection is most often done by measuring increased transport of molecules across the membrane, into or out of the cell. We review here various methods of electroporation detection, together with their advantages and disadvantages. Electroporation detection can be carried out by using dyes (fluorophores or colour stains) or functional molecules, by measuring the efflux of biomolecules, by impedance measurements and voltage clamp techniques as well as by monitoring cell swelling. This review describes methods of detecting cell membrane electroporation in order to help researchers choose the most suitable ones for their specific experiments, considering available equipment and experimental conditions.
In situ Ca<sup>2+</sup> titration in the fluorometric study of intracellular Ca<sup>2+</sup> binding
2014, Cell Calcium
Citation Excerpt :
Fig. 5 clearly demonstrated the saturation of Ca2+ binding sites in pituitary nerve terminals, and this saturation reflected Ca2+ binding to both the fluorescent dye and an endogenous buffer. Quantitative interpretation of Ca2+ fluorometry experiments depends on accurate measurements of the Kd of the Ca2+ indicator, but this quantity can vary between in vitro solutions where it is typically determined versus cellular environments where it is used [4–6,26,30–35]. The difference presumably reflects the influence of the local chemical environment on the Ca2+ binding equilibrium; the Kd is sensitive to pH, ionic strength, temperature, and physiological concentrations of protein [35] (see the Table 19.2 of Molecular Probes Handbook for a number of examples; http://www.lifetechnologies.com).
Imaging with Ca²⁺-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca²⁺ signaling. The spatiotemporal evolution of intracellular free Ca²⁺ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca²⁺ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca²⁺ imaging data, and can be exploited to investigate endogenous Ca²⁺ buffer properties. Here we extended the use of Ca²⁺ fluorometry in the characterization of Ca²⁺ binding molecules within cells, building on a method of titration of intracellular Ca²⁺ binding sites in situ with measured amounts of Ca²⁺ entering through voltage-gated Ca²⁺ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca²⁺ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca²⁺ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca²⁺ buffer and dye, this method yielded estimates of the endogenous buffer concentration and K_d, the dye K_d, and the fraction of Ca²⁺ inaccessible cellular volume. The in situ K_d of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca²⁺-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca²⁺ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca²⁺ binding species. This method offers a versatile approach to the study of endogenous Ca²⁺ binding molecules in their physiological milieu.

View all citing articles on Scopus

View full text

ResearchDetermination of in situ dissociation constant for Fura-2 and quantitation of background fluorescence in astrocyte cell line U373-MG

Abstract

J Biol Chem

Biophys J

Biophys

Biophys J

Biophys J

Biophys J

Biophys J

Methods Cell Biol

Cell Calcium

Cell Calcium

Methods Cell Biol

J Biol Chem

J Biol Chem

Research
Determination of in situ dissociation constant for Fura-2 and quantitation of background fluorescence in astrocyte cell line U373-MG