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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 43-50 |
| Number of pages | 8 |
| Journal | Journal of Neuroscience Methods |
| Volume | 291 |
| DOIs | |
| State | Published - Nov 1 2017 |
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Keywords
- Drug discovery
- High content analysis
- Image processing
- Microglia
- Morphometry
- Phagocytosis
- Phenotypic screening
ASJC Scopus subject areas
- Neuroscience(all)
Cite this
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.
In: Journal of Neuroscience Methods, Vol. 291, 01.11.2017, p. 43-50.Research output: Contribution to journal › Article
}
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 - 2017/11/1
Y1 - 2017/11/1
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
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UR - http://www.scopus.com/inward/citedby.url?scp=85027509724&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2017.08.004
DO - 10.1016/j.jneumeth.2017.08.004
M3 - Article
C2 - 28789994
AN - SCOPUS:85027509724
VL - 291
SP - 43
EP - 50
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
ER -