title: Phagocytosis of apoptotic cells assessed by flow cytometry using 7-Aminoactinomycin D.
abstract: 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.
authors.affiliation: Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain. jgaforio@ujaen.es
authors.forename: J J
authors.initials: JJ
authors.lastname: Gaforio
authors.forename: M J
authors.initials: MJ
authors.lastname: Serrano
authors.forename: I
authors.initials: I
authors.lastname: Algarra
authors.forename: E
authors.initials: E
authors.lastname: Ortega
authors.forename: G
authors.initials: G
authors.lastname: Alvarez de Cienfuegos
mesh_terms: D000818:Animals; D017209:Apoptosis; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D016399:Lymphoma, T-Cell; D051379:Mice; D010587:Phagocytosis; D014407:Tumor Cells, Cultured / Q000166:cytology
chemical_list: D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D