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Quantification of Efferocytosis by Single-cell Fluorescence Microscopy
Published on: August 18, 2018
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Quantification of Efferocytosis by Single-cell Fluorescence Microscopy
Kyle Taruc1, Charles Yin1, Daniel G Wootton2
1Department of Microbiology and Immunology and the Center for Human Immunology, University of Western Ontario.
Journal of Visualized Experiments : Jove
|September 4, 2018
Summary
This study presents a dual-labeling method to accurately quantify efferocytosis (apoptotic cell clearance) by macrophages. The technique distinguishes engulfed from non-engulfed cell fragments, enabling detailed study of efferocytic processes.
Area of Science:
- Immunology
- Cell Biology
Background:
- Efferocytosis, the clearance of apoptotic cells, is crucial for tissue homeostasis and immune regulation.
- Accurate quantification of efferocytosis is challenging due to piecemeal uptake of apoptotic cells.
- Existing methods like flow cytometry struggle to differentiate between internalized and non-internalized cellular material.
Purpose of the Study:
- To develop and validate a novel dual-labeling method for precise quantification of efferocytosis dynamics and capacity.
- To overcome limitations of current techniques in distinguishing efferocytosed versus residual apoptotic cell fragments.
- To enable detailed investigation of cellular signaling pathways involved in efferocytosis.
Main Methods:
- Apoptotic cells are dual-labeled: cytosol with a cell-tracking dye and surface with biotin.
- Macrophages (efferocytes) are analyzed using fluorescent microscopy after incubation with labeled apoptotic cells.
- Internalized versus surface-bound apoptotic material is differentiated using fluorescently labeled streptavidin.
Main Results:
- The dual-labeling method accurately quantifies efferocytic capacity by distinguishing bound from internalized apoptotic cell fractions.
- Live-cell microscopy allows for real-time monitoring of efferocytosis dynamics.
- The approach facilitates the study of efferocytosis in cells expressing fluorescent reporters for signaling pathways.
Conclusions:
- This dual-labeling protocol provides a flexible and accurate method for quantifying efferocytosis.
- It enables precise measurement of efferocytic activity and detailed interrogation of associated cellular signaling.
- The method offers significant advantages over traditional techniques like flow cytometry for studying efferocytosis.
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