FCS experiments to quantify Ca2+ diffusion and its interaction with buffers

Ca2+ signals are ubiquitous. One of the key factors for their versatility is the variety of spatio-temporal distributions that the cytosolic Ca2+ can display. In most cell types Ca2+ signals not only depend on Ca2+ entry from the extracellular medium but also on Ca2+ release from internal stores, a...

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Autores principales:	Sigaut, L., Villarruel, C., Ponce Dawson, S.
Formato:	JOUR
Materias:	Diffusion Enzyme activity Fluorescence Fluorescence spectroscopy Reaction rates Spectroscopic analysis Experimental conditions Extracellular medium Extract informations Fitting parameters Fluorescence Correlation Spectroscopy Optical technique Single wavelength Spatiotemporal distributions Calcium
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_00219606_v146_n10_p_Sigaut
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	todo:paper_00219606_v146_n10_p_Sigaut
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spelling	todo:paper_00219606_v146_n10_p_Sigaut2023-10-03T14:24:36Z FCS experiments to quantify Ca2+ diffusion and its interaction with buffers Sigaut, L. Villarruel, C. Ponce Dawson, S. Diffusion Enzyme activity Fluorescence Fluorescence spectroscopy Reaction rates Spectroscopic analysis Experimental conditions Extracellular medium Extract informations Fitting parameters Fluorescence Correlation Spectroscopy Optical technique Single wavelength Spatiotemporal distributions Calcium Ca2+ signals are ubiquitous. One of the key factors for their versatility is the variety of spatio-temporal distributions that the cytosolic Ca2+ can display. In most cell types Ca2+ signals not only depend on Ca2+ entry from the extracellular medium but also on Ca2+ release from internal stores, a process which is in turn regulated by cytosolic Ca2+ itself. The rate at which Ca2+ is transported, the fraction that is trapped by intracellular buffers, and with what kinetics are thus key features that affect the time and spatial range of action of Ca2+ signals. The quantification of Ca2+ diffusion in intact cells is quite challenging because the transport rates that can be inferred using optical techniques are intricately related to the interaction of Ca2+ with the dye that is used for its observation and with the cellular buffers. In this paper, we introduce an approach that uses Fluorescence Correlation Spectroscopy (FCS) experiments performed at different conditions that in principle allows the quantification of Ca2+ diffusion and of its reaction rates with unobservable (non-fluorescent) Ca2+ buffers. To this end, we develop the necessary theory to interpret the experimental results and then apply it to FCS experiments performed in a set of solutions containing Ca2+, a single wavelength Ca2+ dye, and a non-fluorescent Ca2+ buffer. We show that a judicious choice of the experimental conditions and an adequate interpretation of the fitting parameters can be combined to extract information on the free diffusion coefficient of Ca2+ and of some of the properties of the unobservable buffer. We think that this approach can be applied to other situations, particularly to experiments performed in intact cells. © 2017 Author(s). Fil:Sigaut, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ponce Dawson, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00219606_v146_n10_p_Sigaut
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	Diffusion Enzyme activity Fluorescence Fluorescence spectroscopy Reaction rates Spectroscopic analysis Experimental conditions Extracellular medium Extract informations Fitting parameters Fluorescence Correlation Spectroscopy Optical technique Single wavelength Spatiotemporal distributions Calcium
spellingShingle	Diffusion Enzyme activity Fluorescence Fluorescence spectroscopy Reaction rates Spectroscopic analysis Experimental conditions Extracellular medium Extract informations Fitting parameters Fluorescence Correlation Spectroscopy Optical technique Single wavelength Spatiotemporal distributions Calcium Sigaut, L. Villarruel, C. Ponce Dawson, S. FCS experiments to quantify Ca2+ diffusion and its interaction with buffers
topic_facet	Diffusion Enzyme activity Fluorescence Fluorescence spectroscopy Reaction rates Spectroscopic analysis Experimental conditions Extracellular medium Extract informations Fitting parameters Fluorescence Correlation Spectroscopy Optical technique Single wavelength Spatiotemporal distributions Calcium
description	Ca2+ signals are ubiquitous. One of the key factors for their versatility is the variety of spatio-temporal distributions that the cytosolic Ca2+ can display. In most cell types Ca2+ signals not only depend on Ca2+ entry from the extracellular medium but also on Ca2+ release from internal stores, a process which is in turn regulated by cytosolic Ca2+ itself. The rate at which Ca2+ is transported, the fraction that is trapped by intracellular buffers, and with what kinetics are thus key features that affect the time and spatial range of action of Ca2+ signals. The quantification of Ca2+ diffusion in intact cells is quite challenging because the transport rates that can be inferred using optical techniques are intricately related to the interaction of Ca2+ with the dye that is used for its observation and with the cellular buffers. In this paper, we introduce an approach that uses Fluorescence Correlation Spectroscopy (FCS) experiments performed at different conditions that in principle allows the quantification of Ca2+ diffusion and of its reaction rates with unobservable (non-fluorescent) Ca2+ buffers. To this end, we develop the necessary theory to interpret the experimental results and then apply it to FCS experiments performed in a set of solutions containing Ca2+, a single wavelength Ca2+ dye, and a non-fluorescent Ca2+ buffer. We show that a judicious choice of the experimental conditions and an adequate interpretation of the fitting parameters can be combined to extract information on the free diffusion coefficient of Ca2+ and of some of the properties of the unobservable buffer. We think that this approach can be applied to other situations, particularly to experiments performed in intact cells. © 2017 Author(s).
format	JOUR
author	Sigaut, L. Villarruel, C. Ponce Dawson, S.
author_facet	Sigaut, L. Villarruel, C. Ponce Dawson, S.
author_sort	Sigaut, L.
title	FCS experiments to quantify Ca2+ diffusion and its interaction with buffers
title_short	FCS experiments to quantify Ca2+ diffusion and its interaction with buffers
title_full	FCS experiments to quantify Ca2+ diffusion and its interaction with buffers
title_fullStr	FCS experiments to quantify Ca2+ diffusion and its interaction with buffers
title_full_unstemmed	FCS experiments to quantify Ca2+ diffusion and its interaction with buffers
title_sort	fcs experiments to quantify ca2+ diffusion and its interaction with buffers
url	http://hdl.handle.net/20.500.12110/paper_00219606_v146_n10_p_Sigaut
work_keys_str_mv	AT sigautl fcsexperimentstoquantifyca2diffusionanditsinteractionwithbuffers AT villarruelc fcsexperimentstoquantifyca2diffusionanditsinteractionwithbuffers AT poncedawsons fcsexperimentstoquantifyca2diffusionanditsinteractionwithbuffers
_version_	1807316474806665216

FCS experiments to quantify Ca2+ diffusion and its interaction with buffers

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