Flow cytometric assays for the study of autophagy

Warnes, Gary

doi:10.1016/j.ymeth.2015.03.027

Cited by 41 publications

(30 citation statements)

References 38 publications

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“…Markers of monocytes were mouse anti-human CD282-FITC, CD283-PE, and mouse anti-human CD284-APC. Mouse anti-human LC3B-PE was used as a marker for the autophagy-related protein (28). Intracellular staining was performed using the following mouse anti-human mAb: TNF-α, IL-6, IL-1β, and IL-10.…”

Section: Methodsmentioning

confidence: 99%

Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization through LC3-Dependent Autophagy

Wang

et al. 2017

Front. Immunol.

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Brucellosis is caused by infection with Brucella species and exhibits diverse clinical manifestations in infected humans. Monocytes and macrophages are not only the first line of defense against Brucella infection but also a main reservoir for Brucella. In the present study, we examined the effects of Brucella infection on human peripheral monocytes and monocyte-derived polarized macrophages. We showed that Brucella infection led to an increase in the proportion of CD14++CD16− monocytes and the expression of the autophagy-related protein LC3B, and the effects of Brucella-induced monocytes are inhibited after 6 weeks of antibiotic treatment. Additionally, the production of IL-1β, IL-6, IL-10, and TNF-α from monocytes in patients with brucellosis was suppressed through the LC3-dependent autophagy pathway during Brucella infection. Moreover, Brucella infection inhibited macrophage polarization. Consistently, the addition of 3-MA, an inhibitor of LC3-related autophagy, partially restored macrophage polarization. Intriguingly, we also found that the upregulation of LC3B expression by rapamycin and heat-killed Brucella in vitro inhibits M2 macrophage polarization, which can be reversed partially by 3-MA. Taken together, these findings reveal that Brucella dysregulates monocyte and macrophage polarization through LC3-dependent autophagy. Thus, targeting this pathway may lead to the development of new therapeutics against Brucellosis.

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Section: Methodsmentioning

confidence: 99%

Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization through LC3-Dependent Autophagy

Wang

et al. 2017

Front. Immunol.

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“…The uptake of AO by lysosomes of live cells is a consequence of protonation and entrapment that leads to the accumulation and aggregation of this basic fluorochrome within the acidic organelles. It should also be noted that AO is the key marker of lysosomes used in wideranging studies of autophagy (Demishtein, Porat, Elazar, & Shvets, 2015;SenthilKumar, Skiba, & Kimple, 2019;Thomé et al, 2016;Warnes, 2015). The uptake of AO by lysosomes has therefore been explored as a means of photodynamic-induced cytotoxicity (Zdolsek, Olsson, & Brunk, 1990) for selectively targeting cancer cells (Kusuzaki et al, 2012;Lin et al, 2017).…”

Section: Of 14mentioning

confidence: 99%

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

et al. 2019

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The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G 0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain singleversus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. C 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single-versus double-stranded DNA with acridine orange Keywords: acridine orange r apoptosis r autophagy r cell cycle r lysosomes r mitosis r sperm chromatin fertility assay How to cite this article: Darzynkiewicz, Z., Halicka, D. H., Zhao, H., & Li, J. (2019). Assessment of DNA susceptibility to denaturation as a marker of chromatin structure. BASIC PROTOCOL DNA DenaturationThe process of splitting double-stranded DNA (dsDNA) into single strands, which involves breaking hydrogen bonds between the bases in the DNA double-helical structure, is defined as DNA denaturation. DNA in solution undergoes denaturation when heated, and the temperature at which half remains in double-stranded conformation while the other half is single stranded is called the melting temperature (T m ). Because the G-C base pair, maintained by three hydrogen bonds, is stronger that the two-base pairing of AO has unique metachromatic properties: under the correct conditions (AO concentration, ionic strength, temperature), it can differentially stain ssDNA and dsDNA. Specifically, when AO intercalates between the base pairs of dsDNA, its fluorescence is maximally excited at 480 nm, its emission is at 530 nm, and its excitation lifetime is ß2 ns (Kapuscinski & Darzynkiewicz, 1984Kapuscinski, Darzynkiewicz, & Melamed, 1982. These values reflect singlet excitation of AO. The interaction of AO with ssDNA is more complex: it involves the insertion of the AO cation between adj...

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“…Over the past two decades, research into understanding the molecular basis of autophagy and investigation into its roles in physiology and pathophysiology has intensified. Thus, an understanding of autophagic molecular processes has led to the development of new and innovative tools for rapid and precise detection and quantification of autophagy marker profiles (Degtyarev et al, 2014;Klionsky et al, 2016;Mizushima et al, 2010;Phadwal et al, 2012;Shen et al, 2011;Warnes 2015). Several methods for assessing autophagy have been designed specifically for flow cytometric analysis utilizing autophagic markers (Chan et al, 2012;Klionsky et al, 2016;Phadwal et al, 2012;Warnes, 2015).…”

Section: Autolysosome Maturation and Degradationmentioning

confidence: 99%

“…In addition, flow cytometry has the capability to measure multiple phenotypic markers in a single cell, making it particularly useful for measuring dynamic processes such as autophagic flux. Commonly used and well-characterized autophagosome markers are available, which includes identifying and measuring the levels of autophagosomes, aggresomes, LC3-I turnover, LC3B-II puncta and p62 protein levels Mizushima et al, 2010;Warnes, 2015).…”

Section: Autolysosome Maturation and Degradationmentioning

confidence: 99%

“…The techniques to quantify autophagic markers described in this section are currently being developed to evaluate the role of autophagy in toxicology and monitoring autophagy in clinical samples. The combination of rapid throughput with precise quantification of autophagy within a cell population is also well suited for the development of drug therapies that alter autophagic flux (Warnes, 2015). However, modifications to improve the specificity and sensitivity of these methods are likely to occur with their continued development and implementation.…”

Section: Autolysosome Maturation and Degradationmentioning

confidence: 99%

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Autophagy function and its relationship to pathology, clinical applications, drug metabolism and toxicity

Petibone

Majeed

Casciano

2016

J of Applied Toxicology

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Autophagy is a cellular process that facilitates nutrient turnover and removal of expended macromolecules and organelles to maintain homeostasis. The recycling of cytosolic macromolecules and damaged organelles by autophagosomes occurs through the lysosomal degradation pathway. Autophagy can also be upregulated as a prosurvival pathway in response to stress stimuli such as starvation, hypoxia or cell damage. Over the last two decades, there has been a surge in research revealing the basic molecular mechanisms of autophagy in mammalian cells. A corollary of an advanced understanding of autophagy has been a concurrent expansion of research into understanding autophagic function and dysfunction in pathology. Recent studies have revealed a pivotal role for autophagy in drug toxicity, and for utilizing autophagic components as diagnostic markers and therapeutic targets in treating disease and cancer. In this review, advances in understanding the molecular basis of mammalian autophagy, methods used to induce and evaluate autophagy, and the diverse interactions between autophagy and drug toxicity, disease progression and carcinogenesis are discussed. Copyright © 2016 John Wiley & Sons, Ltd.

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Flow cytometric assays for the study of autophagy

Cited by 41 publications

References 38 publications

Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization through LC3-Dependent Autophagy

Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization through LC3-Dependent Autophagy

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

Autophagy function and its relationship to pathology, clinical applications, drug metabolism and toxicity

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