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Plaque2.0-A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion.

Yakimovich A, Andriasyan V, Witte R, Wang IH, Prasad V, Suomalainen M, Greber UF - PLoS ONE (2015)

Bottom Line: Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene.Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies.Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT
Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.

No MeSH data available.


Related in: MedlinePlus

Cell density dependent features of clonal cancer cell growth in co-cultures with normal cells.(A) Time-lapse analyses of HeLa-H2B-mCherry cell colony formation on WI-38 fibroblasts at sub-confluent (4000 WI-38 cells per well) or confluent density (32000 cells per well). Colony outlines are indicated by dashed green lines. (B) Confocal fluorescence microscopy of HeLa-H2B-mCherry cocultured with 32000, 16000, 8000 or 4000 WI-38 cells (cell numbers determined at seeding time). The samples were fixed 5 days post HeLa cell seeding. Cells were stained with Hoechst nuclear dye (blue) and immuno-stained for ß-catenin (green). Z-sections across the cultures along the dotted lines are shown on the right side and the bottom of the images. (C) Colony phenotypes of HeLa-H2B-mCherry cells co-cultured with WI-38 fibroblasts upon live-imaging and analysis with Plaque2.0. The area of the colonies is a measure of growth cell growth, the eccentricity of the elliptic colony shapes, defined as the ratio of the distance of the elliptic foci to the major axis, serves as an indicator of the cell environment, and the crowdedness fraction is an indicator of the HeLa cell colony density. Mean values from 3 technical replicas containing at least 60 individual colonies per condition are shown, and error bars represent the standard deviations of the respective means. Statistical significance was determined by the Komogorov-Smirnov nonparametric test.
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pone.0138760.g007: Cell density dependent features of clonal cancer cell growth in co-cultures with normal cells.(A) Time-lapse analyses of HeLa-H2B-mCherry cell colony formation on WI-38 fibroblasts at sub-confluent (4000 WI-38 cells per well) or confluent density (32000 cells per well). Colony outlines are indicated by dashed green lines. (B) Confocal fluorescence microscopy of HeLa-H2B-mCherry cocultured with 32000, 16000, 8000 or 4000 WI-38 cells (cell numbers determined at seeding time). The samples were fixed 5 days post HeLa cell seeding. Cells were stained with Hoechst nuclear dye (blue) and immuno-stained for ß-catenin (green). Z-sections across the cultures along the dotted lines are shown on the right side and the bottom of the images. (C) Colony phenotypes of HeLa-H2B-mCherry cells co-cultured with WI-38 fibroblasts upon live-imaging and analysis with Plaque2.0. The area of the colonies is a measure of growth cell growth, the eccentricity of the elliptic colony shapes, defined as the ratio of the distance of the elliptic foci to the major axis, serves as an indicator of the cell environment, and the crowdedness fraction is an indicator of the HeLa cell colony density. Mean values from 3 technical replicas containing at least 60 individual colonies per condition are shown, and error bars represent the standard deviations of the respective means. Statistical significance was determined by the Komogorov-Smirnov nonparametric test.

Mentions: Clonal phenotypes are typical features for viral plaques, and also describe propagation of cancer cells among normal cells. We thus tested if Plaque2.0 detected cancer cell colonies. HeLa cells stably expressing mCherry-tagged histone H2B (HeLa-H2B) were seeded at low concentration among non-labeled normal diploid human lung WI-38 fibroblasts grown to different confluency. Live fluorescence and transmission light microscopy revealed that the shape of the HeLa-H2B colonies was significantly different depending on the confluency of the WI-38 cells (Fig 7A). In case of low WI-38 confluency HeLa-H2B cells were growing in the same Z-plane as WI-38, whereas at high confluency they grew on top of WI-38 cells, as indicated by z-stack confocal microscopy analyses (Fig 7B). We measured the eccentricity of the HeLa-H2B colonies at different WI-38 densities. Eccentricity is defined as the ratio of the distance between the two foci of the ellipse to the length of the major axis, equaling zero for a circle. At low WI-38 density, the HeLa-H2B colonies occupied a larger area and had a lower eccentricity than at high WI-38 density (Fig 7C). At high WI-38 confluence, the shape of HeLa colonies was highly eccentric, unlike at low WI-38 confluence. Plaque2.0 also determined the crowdedness of the colonies, that is the fraction of HeLa-H2B cells within a colony. Interestingly, the crowdedness was largely independent of the WI-38 confluency, although it was significantly higher at the lowest confluency of 4000 WI-38 cells per 96 well compared to 8000. Taken together, the analyses showed that Plaque2.0 is suitable to characterize clonal growth properties of cancer cells. The data demonstrated that HeLa cells preferentially adhered to sites on the dish that are free of WI-38 cells. Particularly highly confluent WI-38 cells slowed down the growth of HeLa colonies.


Plaque2.0-A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion.

Yakimovich A, Andriasyan V, Witte R, Wang IH, Prasad V, Suomalainen M, Greber UF - PLoS ONE (2015)

Cell density dependent features of clonal cancer cell growth in co-cultures with normal cells.(A) Time-lapse analyses of HeLa-H2B-mCherry cell colony formation on WI-38 fibroblasts at sub-confluent (4000 WI-38 cells per well) or confluent density (32000 cells per well). Colony outlines are indicated by dashed green lines. (B) Confocal fluorescence microscopy of HeLa-H2B-mCherry cocultured with 32000, 16000, 8000 or 4000 WI-38 cells (cell numbers determined at seeding time). The samples were fixed 5 days post HeLa cell seeding. Cells were stained with Hoechst nuclear dye (blue) and immuno-stained for ß-catenin (green). Z-sections across the cultures along the dotted lines are shown on the right side and the bottom of the images. (C) Colony phenotypes of HeLa-H2B-mCherry cells co-cultured with WI-38 fibroblasts upon live-imaging and analysis with Plaque2.0. The area of the colonies is a measure of growth cell growth, the eccentricity of the elliptic colony shapes, defined as the ratio of the distance of the elliptic foci to the major axis, serves as an indicator of the cell environment, and the crowdedness fraction is an indicator of the HeLa cell colony density. Mean values from 3 technical replicas containing at least 60 individual colonies per condition are shown, and error bars represent the standard deviations of the respective means. Statistical significance was determined by the Komogorov-Smirnov nonparametric test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0138760.g007: Cell density dependent features of clonal cancer cell growth in co-cultures with normal cells.(A) Time-lapse analyses of HeLa-H2B-mCherry cell colony formation on WI-38 fibroblasts at sub-confluent (4000 WI-38 cells per well) or confluent density (32000 cells per well). Colony outlines are indicated by dashed green lines. (B) Confocal fluorescence microscopy of HeLa-H2B-mCherry cocultured with 32000, 16000, 8000 or 4000 WI-38 cells (cell numbers determined at seeding time). The samples were fixed 5 days post HeLa cell seeding. Cells were stained with Hoechst nuclear dye (blue) and immuno-stained for ß-catenin (green). Z-sections across the cultures along the dotted lines are shown on the right side and the bottom of the images. (C) Colony phenotypes of HeLa-H2B-mCherry cells co-cultured with WI-38 fibroblasts upon live-imaging and analysis with Plaque2.0. The area of the colonies is a measure of growth cell growth, the eccentricity of the elliptic colony shapes, defined as the ratio of the distance of the elliptic foci to the major axis, serves as an indicator of the cell environment, and the crowdedness fraction is an indicator of the HeLa cell colony density. Mean values from 3 technical replicas containing at least 60 individual colonies per condition are shown, and error bars represent the standard deviations of the respective means. Statistical significance was determined by the Komogorov-Smirnov nonparametric test.
Mentions: Clonal phenotypes are typical features for viral plaques, and also describe propagation of cancer cells among normal cells. We thus tested if Plaque2.0 detected cancer cell colonies. HeLa cells stably expressing mCherry-tagged histone H2B (HeLa-H2B) were seeded at low concentration among non-labeled normal diploid human lung WI-38 fibroblasts grown to different confluency. Live fluorescence and transmission light microscopy revealed that the shape of the HeLa-H2B colonies was significantly different depending on the confluency of the WI-38 cells (Fig 7A). In case of low WI-38 confluency HeLa-H2B cells were growing in the same Z-plane as WI-38, whereas at high confluency they grew on top of WI-38 cells, as indicated by z-stack confocal microscopy analyses (Fig 7B). We measured the eccentricity of the HeLa-H2B colonies at different WI-38 densities. Eccentricity is defined as the ratio of the distance between the two foci of the ellipse to the length of the major axis, equaling zero for a circle. At low WI-38 density, the HeLa-H2B colonies occupied a larger area and had a lower eccentricity than at high WI-38 density (Fig 7C). At high WI-38 confluence, the shape of HeLa colonies was highly eccentric, unlike at low WI-38 confluence. Plaque2.0 also determined the crowdedness of the colonies, that is the fraction of HeLa-H2B cells within a colony. Interestingly, the crowdedness was largely independent of the WI-38 confluency, although it was significantly higher at the lowest confluency of 4000 WI-38 cells per 96 well compared to 8000. Taken together, the analyses showed that Plaque2.0 is suitable to characterize clonal growth properties of cancer cells. The data demonstrated that HeLa cells preferentially adhered to sites on the dish that are free of WI-38 cells. Particularly highly confluent WI-38 cells slowed down the growth of HeLa colonies.

Bottom Line: Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene.Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies.Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT
Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.

No MeSH data available.


Related in: MedlinePlus