A more advanced phase of death was observed in some cells as early as 3 h post-infection and in most cells by 18 h.
When caspase-8 is activated in the initial phase of death receptor-induced apoptosis, it can cleave Bid.
This issue might have led to misclassification of phase of death especially for Phase II trauma fatalities with extraordinary long prehospital times.
This presumably reflects previously reported differences in the caspase dependency of the early and late phase of death receptor-induced biphasic JNK activation.
Cells in the early phases of cell death are Annexin V positive and 7AAD-negative, and necrotic cells are Annexin V positive and 7AAD-positive (Figure 2A).
In addition, p27Kip1 was reported to induce caspase -dependent and -independent phases of cell death through TNF-α signaling [ 59].
To quantify the levels of the different phases of cell death, we again used data generated from co-localisation scatter plot analysis, which showed significantly more RGCs in the phase of early apoptosis (P<0.05, green, Anx+) and less necrosis (P<0.05, red, PI+) in 3xTg-AD compared with aged control.
Separation of the different phases of cell death according to whether they were annexin V positive (early apoptosis), annexin V and PI double positive (late apoptosis) or PI positive (necrosis) was enabled by the adoption of thresholds set automatically by the plugin for each analysis.
Data were analysed in a manner similar to that of data derived by in vitro FACS analysis, with four quadrants (midlines shaded in colour overlay as red, green, blue and yellow) enabling separation of the different phases of cell death as annexin V positive (early apoptosis, green), annexin V and PI double positive (late apoptosis, yellow) and PI positive (necrosis, red).
A variety of different TUNEL-positive phenotypes were observed: some cells stained only in perinuclear areas, in others the entire nucleus was strongly positive, and yet others showed a very condensed, pyknotic, TUNEL-positive nucleus, all probably relating to different phases of cell death.
