Abstract
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.
Highlights
In eukaryotes there are two types of cell death, apoptosis and necrosis, that are primarily distinguished by morphology, and differ in biochemical and molecular properties [2,16,23,40,52]
In this study we evaluated and compared the reliability of eight distinct methods (Giemsa staining, trypan blue assay, acridine orange/ethidium bromide double staining for fluorescence microscopy and for flow cytometry, propidium iodide staining, Annexin V, terminal deoxynucleotidyltransferase dUTP nick-end labelling (TUNEL) assay and DNA ladder) for detection and quantification of apoptosis and necrosis in the HL60 human leukaemic cell line exposed to ultraviolet radiation (UV) radiation for different lengths of time, as a model of induction of cell death
Giemsa staining HL60 cells exposed to UV radiation during short periods (3 sec, 15 sec, 30 sec, 1 min and 3 min) showed morphological features of apoptosis in cytospins stained with Giemsa: cell shrinking, chromatin condensation and nuclear fragmentation
Summary
In eukaryotes there are two types of cell death, apoptosis and necrosis, that are primarily distinguished by morphology, and differ in biochemical and molecular properties [2,16,23,40,52]. Necrosis is a pathological form of cell death, resulting in the loss of the cell ability to maintain homeostasis It is morphologically associated with cell swelling, rapid rupture of cytoplasmic, organelle and nuclear membranes, followed by disintegration of organized structures. DNA degradation can occur in necrosis but randomly resulting in a smear pattern in DNA electrophoresis [2,6,23,24, 54,55]. Another biochemical event that was recently shown to be present is loss of plasma membrane asymmetry that occurs early in apoptosis [14,49]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
