Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.
The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.
Leite
M
M
IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Portugal.
Quinta-Costa
M
M
Leite
P S
PS
Guimarães
J E
JE
D000165:Acridine Orange / Q000494:pharmacology; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D001399:Azure Stains / Q000494:pharmacology; D016923:Cell Death / Q000528:radiation effects*; D004396:Coloring Agents / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D005821:Genetic Techniques; D018922:HL-60 Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D007202:Indicators and Reagents / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D009336:Necrosis; D011419:Propidium / Q000494:pharmacology; D013997:Time Factors; D014343:Trypan Blue / Q000494:pharmacology; D014466:Ultraviolet Rays
D017304:Annexin A5; D001399:Azure Stains; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011419:Propidium; D004996:Ethidium; D000165:Acridine Orange; D014343:Trypan Blue