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Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells

Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that t...

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Detalles Bibliográficos
Autores principales: Chan, Leo Li-Ying, Rice, William L., Qiu, Jean
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980413/
https://www.ncbi.nlm.nih.gov/pubmed/31978129
http://dx.doi.org/10.1371/journal.pone.0227950
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author Chan, Leo Li-Ying
Rice, William L.
Qiu, Jean
author_facet Chan, Leo Li-Ying
Rice, William L.
Qiu, Jean
author_sort Chan, Leo Li-Ying
collection PubMed
description Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of the dim and diffuse objects after trypan blue staining. Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrate the co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. The videos clearly show immediate cell rupturing after trypan blue contact. The formation of these diffuse objects was monitored and counted over time as cells die outside of the incubator. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect the outcomes of cellular therapies, which require accurate measurements of immune cells that will be infused back into patients.
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spelling pubmed-69804132020-02-04 Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells Chan, Leo Li-Ying Rice, William L. Qiu, Jean PLoS One Research Article Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of the dim and diffuse objects after trypan blue staining. Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrate the co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. The videos clearly show immediate cell rupturing after trypan blue contact. The formation of these diffuse objects was monitored and counted over time as cells die outside of the incubator. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect the outcomes of cellular therapies, which require accurate measurements of immune cells that will be infused back into patients. Public Library of Science 2020-01-24 /pmc/articles/PMC6980413/ /pubmed/31978129 http://dx.doi.org/10.1371/journal.pone.0227950 Text en © 2020 Chan et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Chan, Leo Li-Ying
Rice, William L.
Qiu, Jean
Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
title Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
title_full Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
title_fullStr Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
title_full_unstemmed Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
title_short Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
title_sort observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980413/
https://www.ncbi.nlm.nih.gov/pubmed/31978129
http://dx.doi.org/10.1371/journal.pone.0227950
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