Hassan Al-Ali1, Han Gao2, Camilla Dalby-Hansen3, Vanessa Ann Peters4, Yan Shi4, Roberta Brambilla5. 1. The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, 33136, USA; Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL, 33136, USA. Electronic address: halali@med.miami.edu. 2. The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, 33136, USA; Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL, 33136, USA. 3. The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, 33136, USA; Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark. 4. The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, 33136, USA. 5. The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, 33136, USA; Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL, 33136, USA. Electronic address: rbrambilla@med.miami.edu.
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.
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.
Authors: Mariana Rinaldi; Laura Thomas; Patricia Mathieu; Pablo Carabias; Maria F Troncoso; Juana M Pasquini; Gabriel A Rabinovich; Laura A Pasquini Journal: Neurobiol Dis Date: 2016-09-06 Impact factor: 5.996
Authors: Minna Yli-Karjanmaa; Bettina Hjelm Clausen; Matilda Degn; Hans Gram Novrup; Ditte Gry Ellman; Peter Toft-Jensen; David E Szymkowski; Allan Stensballe; Morten Meyer; Roberta Brambilla; Kate Lykke Lambertsen Journal: Front Neurosci Date: 2019-08-07 Impact factor: 4.677