An improved method for the detection of DNA fragmentation

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

“…Indeed, the result of the TUNEL assay (Figure 8) provides clear evidence for nuclear DNA fragmentation within frozen-thawed hES cells after 90 min of incubation at 37°C, by which time most of the cells would have lost their viability, as indicated by trypan blue staining ( Figure 5). Fragmentation of nuclear DNA is a well-know hallmark of apoptosis [9,10]. Moreover, immunostaining revealed positive expression of active caspase-3 enzyme (Figure 9), which is another prominent marker of apoptosis [11].…”

“…TUNEL assay for the detection of apoptosisinduced nuclear DNA fragmentation After incubating frozen-thawed hES cells at 37°C for 90 min, the detached non-viable cells were collected by centrifugation and subjected to TUNEL assay to detect the presence of apoptosis-induced DNA fragmentation [9,10]. The assay was carried out with the BD Apoalert TM DNA fragmentation assay kit purchased from BD-Biosiences-Clontech Inc., (Mountain View, CA, USA), according to the recommended instructions of the manufacturer.…”

“…By contrast, if a self-induced apoptotic mechanism is involved, then there would be expected to be a gradual loss of cell viability, as the apoptotic cascade would obviously need time to activate and exert its effect on the cell after freeze-thaw. Moreover, the presence of an apoptotic mechanism would be characterized by fragmentation of nuclear DNA [9], which can easily be detected with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay [10]; as well as expression of active caspase-3 enzyme, which triggers the self-proteolytic cascade within apoptotic cells [11]. Additionally, chromatin condensation and margination to the nuclear membrane, which are the characteristic morphological features of apoptosis [12] can be viewed under transmission electron microscopy.…”

Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma

“…Classical techniques to detect apoptosis used light microscopy to identify morphologic changes; flow cytometry with non-vital dyes to analyse nuclear staining, cell size and DNA ploidy; gel electrophoresis of DNA fragments to visualise a typical DNA 'ladder' resulting from endonuclease cleavage. [11][12][13] These techniques are time-consuming, subjective and do not offer themselves to simple quantitation of apoptotic cells. Although flow cytometry using non-vital dyes is quantifiable, it is based on loss of membrane permeability and is not specific to the apoptotic process.…”

Detection of inducible nitric oxide synthase (iNOS) mRNA by RT-PCR in ATL patients and HTLV-I infected cell lines: clinical features and apoptosis by NOS inhibitor

“…The cells were washed twice with 1 × PBS and analyzed by flow cytometry (FACScan; Becton Dickinson). The fragmented DNA was extracted as previously described, 26 and electrophoresed in 1.5% agarose gels containing 0.5 g/ml ethidium bromide.…”