Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization

Abstract Immunofluorescence is indispensable to monitor redistribution of proteins involved in phagosome–lysosome association pathway-relevant (P–LApr) proteins. The software digitizing the signals of these proteins in an unbiased and automated manner is generally costly and not widely available. Th...

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Autores principales:	Kang Wu, Bo Yan, Douglas B. Lowrie, Tao Li, Xiao-Yong Fan
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/998a6e8c46454e9ca3297ceb9d4cfeae
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spelling	oai:doaj.org-article:998a6e8c46454e9ca3297ceb9d4cfeae2021-12-02T14:12:45ZTailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization10.1038/s41598-020-79425-52045-2322https://doaj.org/article/998a6e8c46454e9ca3297ceb9d4cfeae2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79425-5https://doaj.org/toc/2045-2322Abstract Immunofluorescence is indispensable to monitor redistribution of proteins involved in phagosome–lysosome association pathway-relevant (P–LApr) proteins. The software digitizing the signals of these proteins in an unbiased and automated manner is generally costly and not widely available. The open-source ImageJ plugin EzColocalization, which is for co-localization analysis of reporters in cells, was not straightforward and sufficient for such analysis. We describe here the input of custom Java code in a novel tailored protocol using EzColocalization to digitize the signals of punctum-distributed P–LApr proteins co-localized with phagosomes and to calculate percentages of phagosomes engaged. We showed that SYBR Gold nucleic acid dye could visualize intracellular mycobacteria that did not express a fluorescent protein. This protocol was validated by showing that IFN-γ enhanced the co-localization of a punctum-distributed P–LApr protein (LC3) with Mycobacterium bovis BCG in the monocyte/macrophage-like RAW264.7 cells and that there was greater co-localization of LC3 with BCG than with M. tuberculosis H37Rv in bone marrow-derived macrophages (BMDMs). Although BCG and a derived strain (rBCG-PA) showed a similarly high degree co-localization with LC3 in BMDMs, in RAW264.7 cells BCG showed much less co-localization with LC3 than rBCG-PA indicating the need for caution in interpreting biological significance from studies in cell lines.Kang WuBo YanDouglas B. LowrieTao LiXiao-Yong FanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kang Wu Bo Yan Douglas B. Lowrie Tao Li Xiao-Yong Fan Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization
description	Abstract Immunofluorescence is indispensable to monitor redistribution of proteins involved in phagosome–lysosome association pathway-relevant (P–LApr) proteins. The software digitizing the signals of these proteins in an unbiased and automated manner is generally costly and not widely available. The open-source ImageJ plugin EzColocalization, which is for co-localization analysis of reporters in cells, was not straightforward and sufficient for such analysis. We describe here the input of custom Java code in a novel tailored protocol using EzColocalization to digitize the signals of punctum-distributed P–LApr proteins co-localized with phagosomes and to calculate percentages of phagosomes engaged. We showed that SYBR Gold nucleic acid dye could visualize intracellular mycobacteria that did not express a fluorescent protein. This protocol was validated by showing that IFN-γ enhanced the co-localization of a punctum-distributed P–LApr protein (LC3) with Mycobacterium bovis BCG in the monocyte/macrophage-like RAW264.7 cells and that there was greater co-localization of LC3 with BCG than with M. tuberculosis H37Rv in bone marrow-derived macrophages (BMDMs). Although BCG and a derived strain (rBCG-PA) showed a similarly high degree co-localization with LC3 in BMDMs, in RAW264.7 cells BCG showed much less co-localization with LC3 than rBCG-PA indicating the need for caution in interpreting biological significance from studies in cell lines.
format	article
author	Kang Wu Bo Yan Douglas B. Lowrie Tao Li Xiao-Yong Fan
author_facet	Kang Wu Bo Yan Douglas B. Lowrie Tao Li Xiao-Yong Fan
author_sort	Kang Wu
title	Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization
title_short	Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization
title_full	Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization
title_fullStr	Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization
title_full_unstemmed	Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization
title_sort	tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using imagej plugin ezcolocalization
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/998a6e8c46454e9ca3297ceb9d4cfeae
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Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization

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