title: A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells.
abstract: BACKGROUND
  Human peripheral blood lymphocytes kept in culture after isolation die by an apoptotic process. Detection of apoptosis with labeled Annexin V to demonstrate loss of plasma membrane asymmetry is sensitive, specific, and easy using flow cytometry. This is true in lymphoblastic cell lines when combining Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). However, measurement of apoptosis by flow cytometry in isolated human lymphocytes using Annexin V-FITC/PI is disturbed by the presence of a variable percentage of erythrocytes in the isolated lymphocyte population. To overcome this problem, we have developed and tested a new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells.
  
  
  METHODS
  Peripheral blood lymphocytes are isolated by density gradient centrifugation. Nucleus-containing cells are selected using CD45-phycoerythrin (PE). The lymphocyte subset of interest is selected using CD4, CD8, or CD19 energy-coupled dye (ECD) labeling. Apoptosis is detected using Annexin V-FITC with 7-amino-Actinomycin-D (7-AAD) to distinguish early apoptotic from late apoptotic lymphocytes.
  
  
  RESULTS
  We have developed a new technique to detect apoptosis in isolated human peripheral blood lymphocyte subsets with good reproducibility, coefficient of variation < 17%.
  
  
  CONCLUSIONS
  We now have a validated tool to study apoptosis in subsets of isolated human lymphocytes to increase our knowledge of pathogenesis and therapies in lymphoreticular malignancies.
authors.affiliation: Department of Internal Medicine, Medical Spectrum Twente, Enschede, The Netherlands.
authors.forename: H J
authors.initials: HJ
authors.lastname: Hasper
authors.forename: R M
authors.initials: RM
authors.lastname: Weghorst
authors.forename: D J
authors.initials: DJ
authors.lastname: Richel
authors.forename: J H
authors.initials: JH
authors.lastname: Meerwaldt
authors.forename: F M
authors.initials: FM
authors.lastname: Olthuis
authors.forename: C E
authors.initials: CE
authors.lastname: Schenkeveld
mesh_terms: D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002478:Cells, Cultured; D003116:Color; D004396:Coloring Agents; D003609:Dactinomycin / Q000031:analogs & derivatives; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D016131:Lymphocyte Subsets / Q000737:chemistry / Q000166:cytology*; D011419:Propidium; D015203:Reproducibility of Results
chemical_list: D017304:Annexin A5; D015704:CD4 Antigens; D016827:CD8 Antigens; D004396:Coloring Agents; D005456:Fluorescent Dyes; D003609:Dactinomycin; D011419:Propidium; C025942:7-aminoactinomycin D; D017493:Leukocyte Common Antigens; D016650:Fluorescein-5-isothiocyanate