Comparative analysis of apoptosis in HL60 detected by annexin-V and fluorescein-diacetate

Background: Our aim was to compare and evaluate apoptosis formation as dete cted by propidium-iodide (PI)/annexin-V or PI/fluorescein-diacetate (FDA) a s dose response parameters in a human promyelocytic leukemia cell line, HL6 0. Methods: In exponentially growing HL60 cells, apoptosis was induced by ioni zing radiation, hyperthermia, topotecan, and cytosine beta-D-arabinofuranos ide. At 4 consecutive days following induction, apoptosis was detected by d ouble-labelling, either with PI/annexin-V or PI/FDA. Forward and side scatt er, red (PI), and green (FDA or amnexin-V) fluorescence were measured by fl ow cytometry. Results: While light scatter discriminated between morphologically damaged and undamaged cells, fluorescence differentiated vital, apoptotic, and dead cells. Equal proportions of these three subpopulations were detected by bo th staining techniques. Occasionally, early and mature apoptoses were ident ified as distinct clusters. During the 4-day observation period, no pronoun ced maxims of the apoptotic fractions were obtained with either treatment m odality. The gradual increases usually showed a delay of 1-2 days. Conclusions: FDA and annexin-V are equally suitable for detecting apoptosis . Separation improves with time after induction, indicating that, with resp ect to test specificity, mature apoptoses are superior to early stages. How ever, the sensitivity towards low rates of apoptosis after weak induction a ppears limited with both staining procedures. Cytometry 37:191-196, 1999 (C ) 1999 Wiley-Liss, Inc.