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Measurement of intracellular concentration of fluorescently-labeled targets in living cells

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Item Type:Article
Title:Measurement of intracellular concentration of fluorescently-labeled targets in living cells
Creators Name:Cherkas, V. and Grebenyuk, S. and Osypenko, D. and Dovgan, A.V. and Grushevskyi, E.O. and Yedutenko, M. and Sheremet, Y. and Dromaretsky, A. and Bozhenko, A. and Agashkov, K. and Kononenko, N.I. and Belan, P.
Abstract:Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells.
Keywords:Cytoplasm, Fluorescence Microscopy, Fluorescence Spectrometry, Fluorescent Dyes, Hippocalcin, Hippocampus, Neurons, Animals, Rats
Source:PLoS ONE
ISSN:1932-6203
Publisher:Public Library of Science (U.S.A.)
Volume:13
Number:4
Page Range:e0194031
Date:25 April 2018
Official Publication:https://doi.org/10.1371/journal.pone.0194031
PubMed:View item in PubMed

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