Flow cytometric HyPer-based assay for hydrogen peroxide

Lyublinskaya, O. G.; Antonov, S. A.; Gorokhovtsev, S.G.; Пуговкина, Н. А.; Kornienko, Julia; Ivanova, Ju. S.; Shatrova, A. N.; Aksenov, N. D.; Зенин, В. В.; Nikolsky, Nikolay

doi:10.1016/j.freeradbiomed.2018.05.091

Cited by 14 publications

(13 citation statements)

References 25 publications

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“…Within 10 min after the peroxide addition, at different time points, cell sample aliquots were analyzed with CytoFLEX flow cytometer (Beckman Coulter). HyPer fluorescence in gated HyPer-positive cells was examined by monitoring fluorescence signal at 525 nm registered at 488 nm excitation (hereafter denoted as EX488/FL525 signal), which reflects the accumulation of oxidized form of sensor [10] (Fig. 1 A).…”

Section: Methodsmentioning

confidence: 99%

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

Lyublinskaya

Antunes

2019

Redox Biology

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In this study, we propose a method for quantification of average hydrogen peroxide concentration within a living cell that is based on the use of genetically encoded H 2 O 2 biosensor HyPer. The method utilizes flow cytometric measurements of HyPer fluorescence in H 2 O 2 -exposed cells to analyze the biosensor oxidation kinetics. Fitting the experimental curves with kinetic equations allows determining the rate constants of HyPer oxidation/reduction which are used further for the calculation of peroxide concentrations in the cells of interest both in the presence and absence of external H 2 O 2 . Applying this method to K562 cells, we have estimated the gradient as about 390-fold between the extracellular and intracellular level of exogenous H 2 O 2 in cells exposed to the micromole doses of peroxide, as well as the average basal level of H 2 O 2 in the cytosol of undisturbed cells ( ). The method can be extended to other H 2 O 2 -sensitive redox probes or to procedures in which, rather than adding external peroxide, intracellular production of peroxide is triggered, providing a tool to quantitate not only basal average H 2 O 2 concentrations but also the concentration of peroxide build up in the vicinity of redox probes.

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Section: Methodsmentioning

confidence: 99%

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

Lyublinskaya

Antunes

2019

Redox Biology

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“…In order to extend these observations, we applied genetically encoded hydrogen peroxide (H2O2) biosensor – HyPer (Belousov et al, 2006). The detailed description of this genetic construct as well as its application to estimate intracellular H 2 O 2 levels in ESCs is provided in our previous article (Lyublinskaya et al, 2018). In brief, the intracellular level of oxidizer can be estimated by HyPer fluorescence intensity in young and senescent ESCs stably expressing HyPer.…”

Section: Resultsmentioning

confidence: 99%

“…To assess intracellular H 2 O 2 /pH levels HyPer/SypHer-expressing cells were used. The technical features of these cytometric measurements can be found in the previous studies (Lyublinskaya et al, 2018; Deryabin & Borodkina, in press).…”

Section: Methodsmentioning

confidence: 99%

“…ESCs were transduced with the following lentiviruses: Dec_pPRL-mCherry (Deryabin et al, 2021), cyto-HyPer and SyPher (Lyublinskaya et al, 2018; Deryabin & Borodkina, in press), CRISPR-Cas9-based lentivuruses for SERPINE1 knockout or overexpression. Protocols of lentiviral particles production and ESCs lentiviral transduction are described in detail in our previous article (Deryabin et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

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Senescence of stromal cells contributes to endometrium dysfunction and embryo implantation failure

Deryabin

Ivanova

Borodkina

2021

Preprint

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Successful implantation requires two-sided interaction between genetically normal embryo and properly prepared endometrium. At the cellular level the latter means hormone-regulated differentiation of endometrial stromal cells (ESCs) into decidual ones that create appropriate microenvironment for invading embryo. Impaired decidualization is proved to mediate implantation failures. Here we elicited ESCs’ senescence as the cause for disturbed decidualization of endometrial stroma and impaired blastocyst implantation. Ability to decidualize and to accept modeled blastocysts inversely correlated with senescence in patients’ ESCs lines. Reduced hormonal responsiveness of senescent ESCs led to inappropriate decidualization dynamics resulting in altered receptivity, disturbed ligand-receptor interaction with trophoblasts and modified architecture of extracellular matrix what hindered blastocysts’ invasion. Furthermore, senescent ESCs caused ‘bystander’ quenching of decidual reaction in adjacent cells reinforcing dysfunction of stromal compartment. Implementation of senomorphics reducing senescence phenotype diminished adverse effects of senescent ESCs on decidualization and implantation using both in vitro models and patients’ lines, what suggests a promising strategy to increase in vitro fertilization efficacy.

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“…Nicotinamide nucleotide transhydrogenase (NNT) also regulate H 2 O 2 levels by supporting the thioredoxin and the glutathione antioxidant system (Hanschmann et al, 2013). There is a 100-500-fold gradient of H 2 O 2 concentration between the extracellular and intracellular spaces (Lyublinskaya et al, 2018;Mailloux, 2018;Pak et al, 2020;Sies and Jones, 2020). Control of intracellular H 2 O 2 gradients is maintained mainly by the thioredoxin system and by exchanges between the different cell compartments including the ER, mitochondria, and peroxisomes (Appenzeller-Herzog et al, 2016;Yoboue et al, 2018;Fransen and Lismont, 2019;Mishina et al, 2019).…”

Section: Cytosol Ros and Their Regulation In The Cellmentioning

confidence: 99%

Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man

Sinenko

Starkova

Kuzmin

et al. 2021

Front. Cell Dev. Biol.

132

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Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide, are generated as byproducts of oxidative phosphorylation in the mitochondria or via cell signaling-induced NADPH oxidases in the cytosol. In the recent two decades, a plethora of studies established that elevated ROS levels generated by oxidative eustress are crucial physiological mediators of many cellular and developmental processes. In this review, we discuss the mechanisms of ROS generation and regulation, current understanding of ROS functions in the maintenance of adult and embryonic stem cells, as well as in the process of cell reprogramming to a pluripotent state. Recently discovered cell-non-autonomous ROS functions mediated by growth factors are crucial for controlling cell differentiation and cellular immune response in Drosophila. Importantly, many physiological functions of ROS discovered in Drosophila may allow for deciphering and understanding analogous processes in human, which could potentially lead to the development of novel therapeutic approaches in ROS-associated diseases treatment.

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Flow cytometric HyPer-based assay for hydrogen peroxide

Cited by 14 publications

References 25 publications

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

Senescence of stromal cells contributes to endometrium dysfunction and embryo implantation failure

Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man

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