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Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells
Published on: February 20, 2015
Generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells
Chun Yang1, Wei Xiong2, Qian Qiu2
1Departments of Pediatrics and Pharmacology, University of Montréal.
Abstract:
Interest in the biological roles of cell membrane-derived vesicles in cell-cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 μm to 1 μm, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte-derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.
Insights
Researchers generated T lymphocyte-derived microparticles (LMPs) from apoptotic T cells. These microparticles effectively induce apoptosis in bronchial epithelial cells, offering a new tool for disease and toxicology research.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Cell membrane-derived vesicles, including microparticles (MPs), are increasingly recognized for their roles in intercellular communication.
- Microparticles (MPs) are shed from activated or apoptotic eukaryotic cells and range from 0.1 to 1 μm in diameter.
Purpose of the Study:
- To describe the generation and isolation of T lymphocyte-derived microparticles (LMPs) from apoptotic T cells.
- To characterize LMPs and demonstrate their proapoptotic effects on bronchial epithelial cells.
- To provide a reproducible method for LMP isolation for disease modeling and testing.
Main Methods:
- Generation of LMPs from apoptotic CEM T cells stimulated with actinomycin D.
- Isolation of LMPs using a multistep differential centrifugation process.
- Characterization of LMPs via flow cytometry and assessment of their in situ proapoptotic effects on mouse primary bronchial epithelial cells.
Main Results:
- Successful isolation of abundant quantities of T lymphocyte-derived microparticles (LMPs) from apoptotic lymphocytes in vitro.
- Demonstration of the proapoptotic effect of LMPs on mouse primary bronchial epithelial cells.
- Validation of a reproducible protocol for LMP generation and characterization.
Conclusions:
- The described methods provide a reliable procedure for generating LMPs from apoptotic lymphocytes.
- LMPs can be utilized for evaluating disease models and for pharmacology and toxicology testing.
- The use of primary bronchial tissue explants offers a more relevant model for airway tract investigations compared to immortalized cell lines.
