title: Accurate apoptosis measurement requires quantification of loss of expression of surface antigens and cell fragmentation.
abstract: BACKGROUND
  The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis.
  
  
  METHODS
  Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI).
  
  
  RESULTS
  Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied.
  
  
  CONCLUSIONS
  Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis.
authors.affiliation: CNB-CSIC R&D Associated Unit, Department of Medicine, University of Alcalá, Alcalá de Henares, Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Diaz
authors.forename: Alfredo
authors.initials: A
authors.lastname: Prieto
authors.forename: Hugo
authors.initials: H
authors.lastname: Barcenilla
authors.forename: Jorge
authors.initials: J
authors.lastname: Monserrat
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Sánchez
authors.forename: Eduardo
authors.initials: E
authors.lastname: Reyes
authors.forename: Maria P
authors.initials: MP
authors.lastname: Hernandez-Fuentes
authors.forename: Antonio
authors.initials: A
authors.lastname: de la Hera
authors.forename: Alberto
authors.initials: A
authors.lastname: Orfao
authors.forename: Melchor
authors.initials: M
authors.lastname: Alvarez-Mon
mesh_terms: D000954:Antigens, Surface / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology*; D002452:Cell Count / Q000379:methods*; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology / Q000378:metabolism
chemical_list: D000954:Antigens, Surface