High content analysis of phagocytic activity and cell morphology with PuntoMorph

Hassan Al-Ali*, Han Gao, Camilla Dalby-Hansen, Vanessa Ann Peters, Yan Shi, Roberta Brambilla

*Kontaktforfatter for dette arbejde

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Background Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. New method We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. Results We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. Comparison with existing methods We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. Conclusions We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph.

OriginalsprogEngelsk
TidsskriftJournal of Neuroscience Methods
Vol/bind291
Sider (fra-til)43-50
ISSN0165-0270
DOI
StatusUdgivet - 2017

Fingeraftryk

Phagocytes
Homeostasis
Fluorescence
Maintenance

Citer dette

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title = "High content analysis of phagocytic activity and cell morphology with PuntoMorph",
abstract = "Background Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. New method We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. Results We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. Comparison with existing methods We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. Conclusions We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph.",
keywords = "Drug discovery, High content analysis, Image processing, Microglia, Morphometry, Phagocytosis, Phenotypic screening",
author = "Hassan Al-Ali and Han Gao and Camilla Dalby-Hansen and Peters, {Vanessa Ann} and Yan Shi and Roberta Brambilla",
year = "2017",
doi = "10.1016/j.jneumeth.2017.08.004",
language = "English",
volume = "291",
pages = "43--50",
journal = "Journal of Neuroscience Methods",
issn = "0165-0270",
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High content analysis of phagocytic activity and cell morphology with PuntoMorph. / Al-Ali, Hassan; Gao, Han; Dalby-Hansen, Camilla; Peters, Vanessa Ann; Shi, Yan; Brambilla, Roberta.

I: Journal of Neuroscience Methods, Bind 291, 2017, s. 43-50.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - High content analysis of phagocytic activity and cell morphology with PuntoMorph

AU - Al-Ali, Hassan

AU - Gao, Han

AU - Dalby-Hansen, Camilla

AU - Peters, Vanessa Ann

AU - Shi, Yan

AU - Brambilla, Roberta

PY - 2017

Y1 - 2017

N2 - Background Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. New method We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. Results We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. Comparison with existing methods We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. Conclusions We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph.

AB - Background Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. New method We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. Results We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. Comparison with existing methods We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. Conclusions We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph.

KW - Drug discovery

KW - High content analysis

KW - Image processing

KW - Microglia

KW - Morphometry

KW - Phagocytosis

KW - Phenotypic screening

U2 - 10.1016/j.jneumeth.2017.08.004

DO - 10.1016/j.jneumeth.2017.08.004

M3 - Journal article

VL - 291

SP - 43

EP - 50

JO - Journal of Neuroscience Methods

JF - Journal of Neuroscience Methods

SN - 0165-0270

ER -