MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS

Kauffman, Megan E.; Kauffman, Melinda K.; Traore, Kassim; Zhu, Hong; Trush, Michael A.; Jia, Zhenquan; Li, Y. Robert

doi:10.20455/ros.2016.865

Cited by 146 publications

(142 citation statements)

References 10 publications

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“…As shown in Figure 1, doxorubicin exhibits characteristic fluorescence spectra of excitation and emission with maximum excitation (λex) and emission (λem) wavelengths being 470 and 560 nm, respectively. Notably, the λex and λem are very similar to those of ROS-oxidized MitoSOX [7]. This observation has important implications in using MitoSOX assays to detect the ROS formation induced by doxorubicin.…”

Section: Resultsmentioning

confidence: 96%

“…During our studies on MitoSOX-based fluorescence assays, we observed that doxorubicin has similar fluorescence spectrometric properties to MitoSOX, a probe for detecting reactive oxygen species (ROS), especially superoxide [7]. We thus further characterized the fluorescence properties of doxorubicin in a hope to develop fluorescence-based assays for detecting this commonly used anticancer drug in biological systems.…”

Section: Resultsmentioning

confidence: 99%

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Fluorescence-Based Assays for Measuring Doxorubicin in Biological Systems

Kauffman

Zhu

et al. 2016

ROS

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Detection and measurement of doxorubicin in biological systems, including body fluids, cells, and tissues, are instrumental in understanding the mechanisms of action of this widely used drug in treating cancer as well as in causing adverse effects. In this article, we, for the first time, characterized the use of fluorescence-based techniques, including fluorescence spectrometry, microscopy, and flow cytometry in measuring and/or detecting doxorubicin in biological systems, including cell lysates and cultured intact cells. We showed that doxorubicin has a maximum excitation and emission wavelength of 470 and 560 nm, respectively. The detection sensitivity by fluorescence spectrometry is less than 0.1 μM in buffers and cell lysates. Fluorescence microscopy demonstrated the readily detection of concentration-dependent accumulation of doxorubicin in cultured cells via either green or red fluorescence, but with green fluorescence showing a higher sensitivity of detection. Flow cytometry also revealed sensitive detection of doxorubicin accumulation in cell suspensions in a concentration-dependent manner. The readily and sensitive measurement and detection of doxorubicin by the above three fluorescence-based techniques has important implications in studying the cellular dynamics of doxorubicin in both cancer and normal cells under various experimental conditions.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Fluorescence-Based Assays for Measuring Doxorubicin in Biological Systems

Kauffman

Zhu

et al. 2016

ROS

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“…Cells were washed and stained with Hoechst 33342 stain for 10 minutes at room temperature. For flow cytometry, cells were labeled with dye at 1 µM final concentration, incubated in the dark at 37°C and immediately loaded for ROS fluorescence detection in the FL-2 channel using flow cytometry (Kauffman et al, 2016).…”

Section: Mitochondrial Ros Measurementmentioning

confidence: 99%

Heme synthesis inhibition blocks angiogenesis via mitochondrial dysfunction

Shetty

Sishtla

Park

et al. 2019

Preprint

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The relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe 2+ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis.

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“…Depending on the experiment, cells were either collected immediately for analysis or challenged with H2O2 for an additional 30 minutes prior to collection. Mitochondrial •O2levels were determined with the fluorescent dye, MitoSox Red (Invitrogen), according to an established protocol (Kauffman et al, 2016). Briefly, cells were incubated in fresh media for 4 hours, washed with PBS, and incubated in 1mL of 5µM MitoSOX Red in HBSS (Gibco) for 20 minutes.…”

Section: Flow Cytometry Analyses Of Rosmentioning

confidence: 99%

NNT Regulates Mitochondrial Metabolism in NSCLC Through Maintenance of Fe-S Protein Function

Falzone

et al. 2019

Preprint

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AbstractHuman lung tumors exhibit robust and complex mitochondrial metabolism, likely precipitated by the highly oxygenated nature of pulmonary tissue. As ROS generation is a byproduct of this metabolism, reducing power in the form of nicotinamide adenine dinucleotide phosphate (NADPH) is required to mitigate oxidative stress in response to this heightened mitochondrial activity. Nicotinamide nucleotide transhydrogenase (NNT) is known to sustain mitochondrial antioxidant capacity through the generation of NADPH, however its function in non-small cell lung cancer (NSCLC) has not been established. We found that NNT expression significantly enhances tumor formation and aggressiveness in mouse models of lung tumor initiation and progression. We further show that NNT loss elicits mitochondrial dysfunction independent of substantial increases in oxidative stress, but rather marked by the diminished activities of proteins dependent on resident iron-sulfur clusters. These defects were associated with both NADPH availability and ROS accumulation, suggesting that NNT serves a specific role in mitigating the oxidation of these critical protein cofactors.

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MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS

Cited by 146 publications

References 10 publications

Fluorescence-Based Assays for Measuring Doxorubicin in Biological Systems

Fluorescence-Based Assays for Measuring Doxorubicin in Biological Systems

Heme synthesis inhibition blocks angiogenesis via mitochondrial dysfunction

NNT Regulates Mitochondrial Metabolism in NSCLC Through Maintenance of Fe-S Protein Function

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