Comparative analysis of apoptosis in HL60 detected by annexin-V and fluorescein-diacetate.
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 promyelocytic leukemia cell line, HL60.


METHODS
In exponentially growing HL60 cells, apoptosis was induced by ionizing radiation, hyperthermia, topotecan, and cytosine beta-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.
Bartkowiak
D
D
Department of Radiooncology, University Hospital, Ulm, Germany. detlef.bartkowiak@medizin.uni-ulm.de
Högner
S
S
Baust
H
H
Nothdurft
W
W
Röttinger
E M
EM
D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D002469:Cell Separation; D003561:Cytarabine / Q000494:pharmacology; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism*; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology* / Q000528:radiation effects; D006358:Hot Temperature; D006801:Humans; D011419:Propidium / Q000378:metabolism; D012542:Scattering, Radiation; D019772:Topotecan / Q000494:pharmacology
D017304:Annexin A5; D005452:Fluoresceins; D003561:Cytarabine; D011419:Propidium; D019772:Topotecan; C018506:diacetylfluorescein