Early features of apoptosis detected by four different flow cytometry assays.

Abstract

The objective of this study was to investigate the sensitivity, specificity and reproducibility of some frequently used apoptosis assays. The degree of apoptosis was tested in two T-lymphoblastoid cell lines, HSB and Jurkat, in which apoptosis was induced by ionizing radiation. HSB and Jurkat samples were taken before, and 0, 2, 4, 6, 8 and 24 h after irradiation with 6 and 10 Gray, or with 10 and 14 Gray, respectively. Four frequently used flow cytometric techniques were evaluated: (i) Annexin V/Propidium Iodide assay, detecting the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, simultaneously with preservation of the membrane integrity; (ii) Terminal deoxynucleotidyl Transferase (TdT) Uridine triphosphate (UTP) nick end labelling (TUNEL), revealing the presence of DNA strand breaks; (iii) DNA-flow cytometry, measuring DNA-stainability (DNA-fragmentation assay) and (iv) Phycoerythrin-labelled (PE) Apo2.7-assay, a monoclonal antibody against 7A6 antigen, a protein, which becomes exposed upon the mitochondrial membrane during apoptosis. As a general standard for identifying that apoptosis had occurred, the cells were assessed for the presence of DNA-laddering on agar gel electrophoresis and by demonstration of characteristic cell morphology. Results were as follows: Fluorescein Isothiocyanate (FITC)-labelled Annexin V/Propidium iodide flow cytometry appeared to be the most sensitive, the most specific and the most user-friendly test for measurement of apoptosis of cells in culture conditions in suspension. The expression of 7A6 antigen on the mitochondrial membrane appeared to be not specific for apoptotic cell death.