Flow cytometric HyPer-based assay for hydrogen peroxide.

Autor: Lyublinskaya OG; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia. Electronic address: o.lyublinskaya@mail.ru., Antonov SA; St. Petersburg State Technological Institute (Technical University); Moskovsky pr. 26, St. Petersburg 190013, Russia., Gorokhovtsev SG; St. Petersburg State Technological Institute (Technical University); Moskovsky pr. 26, St. Petersburg 190013, Russia., Pugovkina NA; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia., Kornienko JS; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia., Ivanova JS; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia., Shatrova AN; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia., Aksenov ND; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia., Zenin VV; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia., Nikolsky NN; Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences; Tikhoretsky pr. 4, St. Petersburg 194064, Russia.
Jazyk: angličtina
Zdroj: Free radical biology & medicine [Free Radic Biol Med] 2018 Nov 20; Vol. 128, pp. 40-49. Date of Electronic Publication: 2018 May 31.
DOI: 10.1016/j.freeradbiomed.2018.05.091
Abstrakt: HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H 2 O 2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H 2 O 2 .
(Copyright © 2018 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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