Spectral imaging-based methods for quantifying autophagy and apoptosis

Dolloff, Nathan G.; Ma, Xiahong; Dicker, David T.; Humphreys, Robin; Li, Lin Z.; El‐Deiry, Wafik S.

doi:10.4161/cbt.12.4.17175

Cited by 14 publications

(7 citation statements)

References 24 publications

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“…One possible method to obtain unbiased counting of GFP-LC3 puncta in a large number of cells is to perform multispectral imaging flow cytometry (see Autophagic flux determination using flow and multispectral imaging cytometry). 310 Multispectral imaging flow cytometry allows characterization of single cells within a population by assessing a combination of morphology and immunofluorescence patterns, thereby providing statistically meaningful data. 311 This method can also be used for endogenous LC3, and, therefore, is useful for nontransfected primary cells.…”

Section: A Western Blotting and Ubiquitin-like Protein Conjugation Smentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Klionsky¹,

Abdelmohsen²,

Abe³

et al. 2016

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Section: A Western Blotting and Ubiquitin-like Protein Conjugation Smentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Klionsky¹,

Abdelmohsen²,

Abe³

et al. 2016

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show abstract

“…The percentage of Ki67-positive cells was calculated using a Prism and Reflector Imaging Spectroscopy System (PARISS) hyperspectral imaging system, as described previously. 27 After CD34 staining, 2 independent investigators who were blinded to the identity of the samples scored vessel density as the number of CD34 + vessels present per 20× field (4 fields were counted on 3 separate tumors from the WT and 668E groups).…”

Section: Migration Assaysmentioning

confidence: 99%

CDK1 stabilizes HIF-1α via direct phosphorylation of Ser668 to promote tumor growth

Warfel¹,

Dolloff

Dicker

et al. 2013

Cell Cycle

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“…One possible method to obtain unbiased counting of GFP-LC3 puncta in a large number of cells is to perform multispectral imaging flow cytometry (see Autophagic flux determination using flow and multispectral imaging cytometry). 221 This method can also be used for endogenous LC3, and, therefore, is useful for non-transfected primary cells. 222 Multispectral imaging flow cytometry allows characterization of single cells within a population by assessing a combination of morphology and immunofluorescence patterns, thereby providing statistically meaningful data.…”

mentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Spectral imaging-based methods for quantifying autophagy and apoptosis

Cited by 14 publications

References 24 publications

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

CDK1 stabilizes HIF-1α via direct phosphorylation of Ser668 to promote tumor growth

Guidelines for the use and interpretation of assays for monitoring autophagy

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