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Updated: Nov 15, 2025
In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages
Published on: February 14, 2021
In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages
Hazel Hall-Roberts1, Elena Di Daniel2, William S James3
1James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford; Alzheimer's Research UK Oxford Drug Discovery Institute, Nuffield Department of Medicine Research Building, University of Oxford; UK Dementia Research Institute, Cardiff University; hall-robertsh@cardiff.ac.uk.
Abstract:
Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.
Insights
This study introduces a new in vitro method to observe microglial efferocytosis, the process by which microglia clear dead cells. This technique aids in understanding neurodegenerative diseases like Parkinson's and Alzheimer's.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are key immune cells in the brain, crucial for clearing cellular debris in neurodegenerative diseases.
- Microglial phagocytosis, specifically efferocytosis, can be impaired by genetic and environmental factors.
- Dysfunctional efferocytosis contributes to the pathology of neurodegenerative conditions such as Parkinson's and Alzheimer's disease.
Purpose of the Study:
- To develop a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis.
- To establish a scalable assay for screening potential therapeutic compounds and analyzing genetic variations affecting microglial function.
- To provide a versatile tool for investigating neuroinflammation and cellular clearance mechanisms in neurodegeneration.
Main Methods:
- Utilized an induced pluripotent stem cell (iPSC)-derived microglia model co-cultured with human neuroblastoma cells (SH-SY5Y).
- Labeled neuroblastoma cells with a pH-sensitive dye to track phagocytic cargo after efferocytosis.
- Employed a 96-well plate assay compatible with live-cell time-lapse imaging and fixed-cell high-content microscopy for quantification.
Main Results:
- Successfully generated a high yield of dead neuroblastoma cells displaying phosphatidylserine 'eat-me' signals.
- The protocol allows for both live imaging and robust quantification of efferocytosis via high-content microscopy.
- Demonstrated the assay's suitability for high-throughput screening of small molecule inhibitors and genetic variant analysis.
Conclusions:
- The developed protocol offers a straightforward and efficient method for assessing microglial efferocytosis in vitro.
- This assay is adaptable for various cargoes (e.g., synaptosomes, myelin) and phagocytic cell types, broadening its application in neurodegeneration research.
- The protocol facilitates the study of factors influencing microglial function and the development of targeted neuroprotective strategies.
