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Updated: Aug 18, 2026
Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos
Published on: August 3, 2017
Phagocytosis of apoptotic cells assessed by flow cytometry using 7-Aminoactinomycin D
J J Gaforio1, M J Serrano, I Algarra
1Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain. jgaforio@ujaen.es
Background:
Apoptotic cells are recognized specifically by macrophages and are cleared rapidly by phagocytosis. However, the recognition mechanisms involved in the clearance of apoptotic cells by macrophages are still not fully understood. Therefore, new methods must be designed to better our understanding of the mechanisms of interaction between macrophages and apoptotic cells. 7-Aminoactinomycin D (7-AAD) is a fluorescent DNA-binding stain usually used as a single agent to detect apoptotic cells by flow cytometry. We propose the use of 7-AAD-stained apoptotic cells as targets for a new flow cytometry phagocytosis assay.
Methods:
Murine T-cell lymphoma YAC-1 cells were treated with etoposide to induce apoptosis. Etoposide-treated YAC-1 target cells were stained subsequently with 7-AAD and then coincubated with resident peritoneal macrophages to allow phagocytosis. The samples were analyzed by flow cytometry. Macrophages that had phagocytosed 7-AAD-stained apoptotic cells were identified by their bright red fluorescence and the resulting values were expressed as the percentage of cells.
Results:
The phagocytic cells appeared as a distinct population characterized by bright fluorescence, which could not be detected in the negative controls. The effects of a phagocytic enhancer (interferon-gamma [IFN-gamma]) or inhibitor (incubation at 4 degrees C) were assessed accurately with this flow cytometric method.
Conclusions:
We describe the use of 7-AAD in an assay that is easy and quick to perform. This flow cytometric-based assay allows the quantification of phagocytosis of apoptotic cells by macrophages.
Insights
This study introduces a new flow cytometry assay using 7-Aminoactinomycin D (7-AAD) stained apoptotic cells to quantify macrophage phagocytosis. The method accurately measures the clearance of apoptotic cells, aiding research into immune cell interactions.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Macrophages efficiently clear apoptotic cells via phagocytosis, but recognition mechanisms require further elucidation.
- Understanding macrophage-apoptotic cell interactions is crucial for advancing immunology and disease research.
- 7-Aminoactinomycin D (7-AAD) is a fluorescent stain for detecting apoptotic cells, typically used in flow cytometry.
Purpose of the Study:
- To develop and validate a novel flow cytometry-based assay for quantifying macrophage phagocytosis of apoptotic cells.
- To utilize 7-AAD-stained apoptotic cells as targets in a phagocytosis assay.
- To improve the understanding of macrophage-mediated clearance of cellular debris.
Main Methods:
- Murine T-cell lymphoma YAC-1 cells were induced into apoptosis using etoposide.
- Apoptotic YAC-1 cells were stained with 7-AAD and coincubated with macrophages.
- Phagocytosis was analyzed via flow cytometry, identifying macrophages with bright red fluorescence from ingested cells.
Main Results:
- A distinct population of phagocytic cells exhibiting bright fluorescence was identified, absent in negative controls.
- The assay accurately detected the effects of phagocytosis enhancers like interferon-gamma (IFN-gamma) and inhibitors like cold incubation.
- The flow cytometric method provided quantifiable data on phagocytic activity.
Conclusions:
- A simple, rapid flow cytometry assay using 7-AAD-stained apoptotic cells for quantifying macrophage phagocytosis has been established.
- This assay facilitates the study of macrophage phagocytic function.
- The method offers a valuable tool for immunological research and drug screening.
