EARLY FEATURES OF APOPTOSIS DETECTED BY 4 DIFFERENT FLOW-CYTOMETRY ASSAYS

The objective of this study was to investigate the sensitivity, specif icity and reproducibility of some frequently used apoptosis assays. Th e degree of apoptosis was tested in two T-lymphoblastoid cell lines, H SB and Jurkat, in which apoptosis was induced by ionizing radiation. H SB and Jurkat samples were taken before, and 0, 2, 4, 6, 8 and 24 h af ter irradiation with 6 and 10 Gray, or with 10 and 14 Gray, respective ly. Four frequently used flow cytometric techniques were evaluated: (i ) Annexin V/Propidium lodide assay, detecting the translocation of pho sphatidylserine to the outer leaflet of the plasma membrane, simultane ously with preservation of the membrane integrity; (ii) Terminal deoxy nucleotidyl Transferase (TdT) Uridine triphosphate (UTP) nick end labe lling (TUNEL), revealing the presence of DNA strand breaks; (iii) DNA- flow cytometry, measuring DNA-stainability (DNA-fragmentation assay) a nd (iv) Phycoerythrin-labelled (PE) Apo2.7-assay, a monoclonal antibod y against 7A6 antigen, a protein, which becomes exposed upon the mitoc hondrial membrane during apoptosis, As a general standard for identify ing that apoptosis had occurred, the cells were assessed for the prese nce of DNA-laddering on agar gel electrophoresis and by demonstration of characteristic cell morphology, Results were as follows: Fluorescei n Isothiocyanate (FITC)-labelled Annexin V/Propidium iodide flow cytom etry appeared to be the most sensitive, the most specific and the most user-friendly test for measurement of apoptosis of cells in culture c onditions in suspension. The expression of 7A6 antigen on the mitochon drial membrane appeared to be not specific for apoptotic cell death.