Comparative analysis of apoptosis in HL60 detected by annexin‐V and fluorescein‐diacetate
1999
Background: Our aim was to compare and evaluate apoptosis formation as detected by
propidium-iodide(PI)/
annexin-V or PI/fluorescein-diacetate (FDA) as dose-response parameters in a human
promyelocyticleukemia cell line,
HL60. Methods: In exponentially growing
HL60cells, apoptosis was induced by ionizing radiation, hyperthermia,
topotecan, and cytosine β-D-arabinofuranoside. At 4 consecutive days following induction, apoptosis was detected by double-labelling, either with PI/
annexin-V or PI/FDA. Forward and side scatter, red (PI), and green (FDA or
annexin-V) fluorescence were measured by flow 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 both staining techniques. Occasionally, early and mature apoptoses were identified as distinct clusters. During the 4-day observation period, no pronounced maxima of the apoptotic fractions were obtained with either treatment modality. 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 respect to test specificity, mature apoptoses are superior to early stages. However, the sensitivity towards low rates of apoptosis after weak induction appears limited with both staining procedures. Cytometry 37:191–196, 1999. © 1999 Wiley-Liss, Inc.
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