The assay has been used on the Jurkat T-cell line in published work to date and unexpected findings amongst a plethora of data are that dead cells have a similar phenotype (Live-Dead^+ve/RIP3^+ve/-ve/Caspase-3^+ve/-ve) no matter what form of RCD was induced [5,7-9]. So it is worth reflecting upon the relative incidence of live and dead cells in studies employing immunoblotting techniques. So it is now possible with flow cytometry more than any other technique to identify a range of RCDs including necroptosis, apoptosis, autophagy, ER stress, DNA Damage, hyperactivated PARP, cleaved PARP and parthanatos all within the same sample after induction of cell death by a single drug for example. This methodology strikingly has made it possible to show that autophagic cells have less DNA Damage than non-autophagic cells thus highlighting a mechanism by which autophagy can preserve cell health [8]. Recent drug research at the Blizard Institute highlights the fact that a single cytotoxic drug can induce multiple forms of RCD with induction of autophagy, ER stress and necroptosis in keratinocyte cell lines (personal observation). Current research at Queen Mary University employing heterogeneous cell populations is also underway to investigate how gd T-cells die in response to fungal infection and the associated RCD and ACD DAMPS (Damage Associated Molecular Patterns) signals from the gut tissue in a diseased state [1,13-15]. The role of leucocyte cell death in trauma patient’s survival is similarly under study [1,13-15]. It can be envisaged that this type of flow cytometric approach to the simultaneous measurement of multiple forms of RCD can be broadened to investigate their role in heterogeneous preparation of cells present in other disease states including infection (e.g. HIV), autoimmunity, cardiovascular, nervous and respiratory diseases.

The use of MitoTracker probes has been to date misused by the lack of use of cell viability probes when analysing the effects of cytotoxic drugs [16,17]. Dead cells do not have mitochondrial function and so should be excluded; this has led to misleading conclusions in some respects about the nature of the role of mitochondria in RCDs [16,17]. However recent work in this laboratory making use of cell viability dyes and an active caspase fluorescent substrate along with MitoTracker and ROS probes has shown that live cells after induction of necroptosis (e.g. by shikonin) have hyper-polarized mitochondrial membrane potential with increased ROS levels (manuscript in preparation). I have also previously reported these types of findings with other drugs such as staurosporine, rapamycin and UV-irradiation [18,19]. The results of such an approach to the detection and measurement of RCDs induced by etoposide, shikonin and thapsigargin is beginning to show indeed the complexity of such RCD responses to the action of cytotoxic drugs [8,20]. Recent publications have shown that multiple forms of necroptotic, apoptotic, oncotic and undefined (DN) cells can be defined by their level of gH2AX, PARP, parthanatos, ROS and mitochondrial function which can be modulated by drugs and their inhibitors for example [8,20].

Shortcomings of the assay besides locating permeability resistant MitoTracker and ROS probes would also include the additional use of a RIP1 antibody. This would then allow the specific detection of RIP1-dependent apoptosis rather than by the current indirect approach by using RIP3 which assumes that this molecule is always associated with RIP1. Previous studies employing shikonin or TNFa to induce necroptosis with blockade by necrostatin-1 showed that necroptosis was indeed inhibited but the incidence of RIP1-dependent apoptosis was increased rather than reduced as would be expected [5,7,8]. Work employing a non-phosphorylated MLKL antibody (rather than the specific phosphorylated form, which is not available) has shown no up-regulation of this specific biological marker of necroptosis (personal observation). So the development of these missing antibodies would hopefully improve the current assay as would the inclusion of the biological marker for gasdermin for the detection of pyroptosis rather than activated caspase-1 [1].

Future work would include the addition of a mechanism for the detection of ferroptosis, lysosomal-dependent, immunogenic, MPT driven cell and endtotic deaths. While NETosis appears to be mainly cell type specific for which a flow cytometric methodology has already been developed [1,21]. The phenotyping of the biological markers for each form of RCD so far developed has also recently been applied to Accidental Cell Death (ACD) [9]. In which it has been shown that dead oncotic (or necrotic) cells can be phenotyped to be either RIP3^+ve/-ve whilst being Caspase-3^-ve and were shown to be a DNA Damage phenotype (gH2AX^+ve/PARP^-ve), parthanatos (gH2AX^+ve/PARP^+ve), cleaved PARP (gH2AX^-ve/PARP^+ve) or a DN phenotype (gH2AX^-ve/PARP^-ve), respectively [9]. This has allowed the identification of eight forms of oncosis whose incidence interestingly could be modulated by inhibitors of necroptosis and apoptosis which perhaps indicates the mechanistic signalling routes involved in ACD [9]. This approach to the simultaneous measurement of multiple forms of RCD and ACD expressed in heterogeneous cell populations could also be transferred to tissues by the use of heavy metal antibody labels rather than fluorescence. Thus enabling the use of Hyperion Imaging CyTOF technology to broaden the concept of this type of approach to the measurement of RCD still further. This would allow the detection and analysis of multiple forms of RCD from relatively low numbers of ex vivo cells isolated from tissue and within in situ sections of tissue in the numerous diseases states were a role for a form of cell death is known. This would include for example RCDs occurring in carcinomas, inflammation of gut tissue [13], neurodegenerative diseases [22], Gaucher’s disease [23], ischemia-reperfusion injury [14], liver disease [23] and the targeting of drugs for the treatment of these diseases [24]. This type of approach to gastroenterological diseases and trauma is being undertaken at Queen Mary University and is hoped to reveal cellular mechanisms involved with a clarity not observed before that could aid the treatment of such diseases and may lead to the development of new approaches to their treatment in the near future.

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