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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

Time-lapse analyses of plaque phenotypes from VACV WR and IHD-J strains demonstrate contribution of cell-free virus to spreading.(A) Still micrographs of representative plaque phenotypes from VACV-IHD-J-E/L-GFP and VACV-WR-GFP in either gelled or liquid medium. (B) Example of VACV-WR-E/L-GFP live microscopy plaques (24 h pi) analyzed by Plaque2.0 software. Here superimposed on the micrograph from the GFP signal, green colored pixels designate foreground pixels detected by thresholding, colored line designates plaque borders and red spots highlight local intensity maxima. This procedure allows detection of adjacent plaques. (C) The relative plaque area normalized to the plaque area from gelled medium was plotted as a function of time. Note that VACV-IHD-J plaques occupy a larger area than VACV-WR at late stages of infection owing to cell-free EEV. Results represent averages from over 50 plaques for each condition from 8 technical replicas.
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pone.0138760.g003: Time-lapse analyses of plaque phenotypes from VACV WR and IHD-J strains demonstrate contribution of cell-free virus to spreading.(A) Still micrographs of representative plaque phenotypes from VACV-IHD-J-E/L-GFP and VACV-WR-GFP in either gelled or liquid medium. (B) Example of VACV-WR-E/L-GFP live microscopy plaques (24 h pi) analyzed by Plaque2.0 software. Here superimposed on the micrograph from the GFP signal, green colored pixels designate foreground pixels detected by thresholding, colored line designates plaque borders and red spots highlight local intensity maxima. This procedure allows detection of adjacent plaques. (C) The relative plaque area normalized to the plaque area from gelled medium was plotted as a function of time. Note that VACV-IHD-J plaques occupy a larger area than VACV-WR at late stages of infection owing to cell-free EEV. Results represent averages from over 50 plaques for each condition from 8 technical replicas.

Mentions: We investigated the differences in plaque phenotypes of VACV-IHDJ-E/L-GFP and VACV-WR-E/L-GFP infections. While the IHD-J strain yielded elongated comet-like plaques in liquid medium but not gelled medium, the WR strain gave spherical plaques under both conditions (Fig 3A). Similar to the results in Fig 1, the IHD-J comets contained spherical satellite plaques, unlike the WR strain, suggesting that they arise from mixed cell-free and cell-cell virus transmission events (Fig 3A and 3B).


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)

Time-lapse analyses of plaque phenotypes from VACV WR and IHD-J strains demonstrate contribution of cell-free virus to spreading.(A) Still micrographs of representative plaque phenotypes from VACV-IHD-J-E/L-GFP and VACV-WR-GFP in either gelled or liquid medium. (B) Example of VACV-WR-E/L-GFP live microscopy plaques (24 h pi) analyzed by Plaque2.0 software. Here superimposed on the micrograph from the GFP signal, green colored pixels designate foreground pixels detected by thresholding, colored line designates plaque borders and red spots highlight local intensity maxima. This procedure allows detection of adjacent plaques. (C) The relative plaque area normalized to the plaque area from gelled medium was plotted as a function of time. Note that VACV-IHD-J plaques occupy a larger area than VACV-WR at late stages of infection owing to cell-free EEV. Results represent averages from over 50 plaques for each condition from 8 technical replicas.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587671&req=5

pone.0138760.g003: Time-lapse analyses of plaque phenotypes from VACV WR and IHD-J strains demonstrate contribution of cell-free virus to spreading.(A) Still micrographs of representative plaque phenotypes from VACV-IHD-J-E/L-GFP and VACV-WR-GFP in either gelled or liquid medium. (B) Example of VACV-WR-E/L-GFP live microscopy plaques (24 h pi) analyzed by Plaque2.0 software. Here superimposed on the micrograph from the GFP signal, green colored pixels designate foreground pixels detected by thresholding, colored line designates plaque borders and red spots highlight local intensity maxima. This procedure allows detection of adjacent plaques. (C) The relative plaque area normalized to the plaque area from gelled medium was plotted as a function of time. Note that VACV-IHD-J plaques occupy a larger area than VACV-WR at late stages of infection owing to cell-free EEV. Results represent averages from over 50 plaques for each condition from 8 technical replicas.
Mentions: We investigated the differences in plaque phenotypes of VACV-IHDJ-E/L-GFP and VACV-WR-E/L-GFP infections. While the IHD-J strain yielded elongated comet-like plaques in liquid medium but not gelled medium, the WR strain gave spherical plaques under both conditions (Fig 3A). Similar to the results in Fig 1, the IHD-J comets contained spherical satellite plaques, unlike the WR strain, suggesting that they arise from mixed cell-free and cell-cell virus transmission events (Fig 3A and 3B).

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