Protocol for in vitro phagocytosis assay for primary mouse microglia and human embryonic stem cell-derived microglia
Beika Zhu1, Alicia L Thurber1, Xianhua Piao2
1Weill Institute for Neuroscience, University of California, San Francisco, San Francisco, CA 94158, USA.
Abstract:
Microglia, the resident immune cells of the brain, maintain brain health by clearing detrimental debris, including amyloid-β (Aβ). Here, we present a protocol for assessing Aβ uptake by primary mouse microglia and human embryonic stem cell-derived microglia using an in vitro phagocytosis assay. We describe procedures for pHrodo-labeled oligomeric Aβ treatment and real-time signal detection using the Incucyte SX5 system. We also detail steps for microglia culture, Aβ labeling, and quantification of phagocytosis over time. For complete details on the use and execution of this protocol, please refer to Zhu et al.1.
Insights
This study details a new protocol to measure how well microglia, the brain's immune cells, engulf amyloid-beta (Aβ) peptides. This method helps researchers understand brain health and neurodegenerative diseases.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are crucial for brain health, clearing waste like amyloid-beta (Aβ).
- Dysfunctional microglia contribute to neurodegenerative diseases.
- Assessing Aβ clearance by microglia is vital for understanding disease mechanisms.
Purpose of the Study:
- To present a standardized protocol for quantifying Aβ phagocytosis by microglia.
- To enable real-time assessment of Aβ uptake in primary and stem cell-derived microglia.
- To provide a reproducible method for neuroinflammation research.
Main Methods:
- Utilizing an in vitro phagocytosis assay with pHrodo-labeled oligomeric Aβ.
- Employing the Incucyte SX5 system for real-time signal detection.
- Describing microglia culture, Aβ labeling, and phagocytosis quantification procedures.
Main Results:
- The protocol allows for quantitative assessment of Aβ phagocytosis over time.
- It is applicable to both primary mouse microglia and human iPSC-derived microglia.
- The method provides reliable data for studying microglial function.
Conclusions:
- This protocol offers a robust tool for investigating microglial phagocytic function in vitro.
- It facilitates research into the role of microglia in Aβ clearance and neurodegeneration.
- The standardized method aids in the development of therapeutic strategies targeting microglial function.
