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A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells

PURPOSE: The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. METHODS: Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of...

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Autores principales: Wensley, Harrison J., Johnston, David A., Smith, Wendy S., Holmes, Suzanne E., Flavell, Sopsamorn U., Flavell, David J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928089/
https://www.ncbi.nlm.nih.gov/pubmed/31873810
http://dx.doi.org/10.1007/s11095-019-2725-1
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author Wensley, Harrison J.
Johnston, David A.
Smith, Wendy S.
Holmes, Suzanne E.
Flavell, Sopsamorn U.
Flavell, David J.
author_facet Wensley, Harrison J.
Johnston, David A.
Smith, Wendy S.
Holmes, Suzanne E.
Flavell, Sopsamorn U.
Flavell, David J.
author_sort Wensley, Harrison J.
collection PubMed
description PURPOSE: The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. METHODS: Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. RESULTS: Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity. CONCLUSIONS: Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11095-019-2725-1) contains supplementary material, which is available to authorized users.
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spelling pubmed-69280892020-01-21 A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells Wensley, Harrison J. Johnston, David A. Smith, Wendy S. Holmes, Suzanne E. Flavell, Sopsamorn U. Flavell, David J. Pharm Res Research Paper PURPOSE: The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. METHODS: Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. RESULTS: Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity. CONCLUSIONS: Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11095-019-2725-1) contains supplementary material, which is available to authorized users. Springer US 2019-12-23 2020 /pmc/articles/PMC6928089/ /pubmed/31873810 http://dx.doi.org/10.1007/s11095-019-2725-1 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Research Paper
Wensley, Harrison J.
Johnston, David A.
Smith, Wendy S.
Holmes, Suzanne E.
Flavell, Sopsamorn U.
Flavell, David J.
A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells
title A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells
title_full A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells
title_fullStr A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells
title_full_unstemmed A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells
title_short A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells
title_sort flow cytometric method to quantify the endosomal escape of a protein toxin to the cytosol of target cells
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928089/
https://www.ncbi.nlm.nih.gov/pubmed/31873810
http://dx.doi.org/10.1007/s11095-019-2725-1
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