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23 pages, 10019 KiB

Open AccessArticle

FLIM for Evaluation of Difference in Metabolic Status between Native and Differentiated from iPSCs Dermal Papilla Cells

by Alena Kashirina, Alena Gavrina, Artem Mozherov, Dmitriy Kozlov, Daria Kuznetsova, Ekaterina Vorotelyak, Elena Zagaynova, Ekaterina Kalabusheva and Aleksandra Kashina

Cells 2022, 11(17), 2730; https://doi.org/10.3390/cells11172730 - 1 Sep 2022

Cited by 1 | Viewed by 2091

Abstract

iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells. Full article

(This article belongs to the Section Cellular Metabolism)

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20 pages, 8624 KiB

Open AccessArticle

Energy Metabolism and Intracellular pH Alteration in Neural Spheroids Carrying Down Syndrome

by Alena Kashirina, Alena Gavrina, Emil Kryukov, Vadim Elagin, Yuliya Kolesova, Alexander Artyuhov, Ekaterina Momotyuk, Vepa Abdyyev, Natalia Meshcheryakova, Elena Zagaynova, Erdem Dashinimaev and Aleksandra Kashina

Biomedicines 2021, 9(11), 1741; https://doi.org/10.3390/biomedicines9111741 - 22 Nov 2021

Cited by 6 | Viewed by 3712

Abstract

Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects. Full article

(This article belongs to the Section Neurobiology and Clinical Neuroscience)

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11 pages, 1687 KiB

Open AccessArticle

FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor

by Alexander S. Goryashchenko, Alexey A. Pakhomov, Anastasia V. Ryabova, Igor D. Romanishkin, Eugene G. Maksimov, Alexander N. Orsa, Oxana V. Serova, Andrey A. Mozhaev, Margarita A. Maksimova, Vladimir I. Martynov, Alexander G. Petrenko and Igor E. Deyev

Biosensors 2021, 11(9), 340; https://doi.org/10.3390/bios11090340 - 15 Sep 2021

Cited by 21 | Viewed by 5168

Abstract

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems. Full article

(This article belongs to the Special Issue Advances of Genetically Encoded Biosensors)

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1 pages, 162 KiB

Open AccessAbstract

FLIM Indicators for Quantitative Measurement of pH

by Tatiana R. Simonyan, Anastasia V. Mamontova, Konstantin A. Lukyanov and Alexey M. Bogdanov

Eng. Proc. 2021, 6(1), 33; https://doi.org/10.3390/I3S2021Dresden-10144 - 17 May 2021

Viewed by 1018

Abstract

Monitoring of intracellular pH changes in situ can provide valuable information about cellular metabolism and a deeper understanding of physiological processes. Most traditional fluorescent indicators are only capable of a relative assessment of changes in the studied parameter in the cell. We associate the possibility of measuring the absolute values that characterize the analyte with the detection of the indicator signal in the time domain, where its quantitative measure is the fluorescence lifetime (tau). In this project, we test promising pH-sensitive fluorophores with labile fluorescence lifetimes—EYFP-G65T and EGFP-Y145L/S205V—both as fluorescent cores for the previously described pH indicators and as independent pH indicators. Measurement of the fluorescence attenuation kinetics of four structures (EYFP-G65T, EGFP-Y145L/S205V, SypHer3s, and SypHer3s-G65T) over a wide pH range revealed areas where tau is linearly dependent on pH. The differences in the fluorescence excitation modes of these molecules makes it possible to use them in one experimental system to assess pH changes in a wide range, 4.0–9.0. We showed that under the conditions of traditional fluorescence microscopy (in the cytoplasm of HEK293 cells), the SypHer3s-G65T indicator shows a dynamic response range approximately 3 times wider than the original SypHer3s. Full article

(This article belongs to the Proceedings of The 8th International Symposium on Sensor Science)

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