Abstract
•We describe the development of a new algorithm, PuntoMorph, for automated counting of phagocytosed beads in 2D images.•PuntoMorph simultaneously analyzes cell morphology on the same images.•PuntoMorph consistently outperforms automated and manual counting methods.•PuntoMorph includes additional modules for high content analysis, making it a standalone option for phenotypic screening.•The combination of the assay system and analytical algorithm is a powerful tool for drug discovery.
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.
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.
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.
We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution.
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.
