Generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells.

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.