Flow cytometric detection of hyper-polarized mitochondria in regulated and accidental cell death processes.

Shikonin induced necroptosis in Jurkat cells were identified flow cytometrically by the up-regulation of RIP3 in live cells and that a proportion of these cells underwent other forms of regulated cell death (RCD) which included parthanatos (< 10%), or cleaved PARP (< 10%) and DNA Damage (> 30%). Live necroptotic cells also possessed functioning mitochondria with hyper-polarized mitochondria membrane potential and generated a fivefold increase in cellular reactive oxygen species (ROS) which was resistant to inhibition by zVAD and necrostatin-1 (Nec-1). After loss of plasma membrane integrity these dead necroptotic cells then showed a higher incidence of parthanatos (> 40%), or cleaved PARP (> 15%) but less DNA Damage (< 15%). Inhibition of shikonin induced apoptosis and necroptosis by zVAD and Nec-1 respectively resulted in live necroptotic cells with an increased incidence of cleaved PARP and reduced levels of DNA Damage respectively. Dead necroptotic cells then showed a reduced incidence of parthanatos and DNA Damage after inhibition by zVAD and Nec-1 respectively. A high proportion of these dead necroptotic cells (30%) which lacked plasma membrane integrity also displayed functioning hyper-polarized mitochondria with high levels of cellular ROS and thus had the capacity to influence the outcome of RCD processes rather than just been the end product of cell death, the necrotic cell. Flow cytometry can thus measure multiple forms of RCD and the level of cellular ROS and MMP which highlights the inter-connection between cell death processes and that a single cell may simultaneously display multiple forms of RCD.

Simultaneous polychromatic flow cytometric detection of multiple forms of regulated cell death.

Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3+ ve/RIP3- ve), necroptosis (RIP3Hi + ve/Caspase-3- ve) and RIP1-dependent apoptosis (Caspase-3+ ve/RIP3+ ve) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations. Shikonin simultaneously induced Jurkat cell apoptosis and necroptosis the degree of which can be shown flow cytometrically together with the effects of blockade of these forms of cell death by zVAD and necrostatin-1 have on specific RCD populations including necroptosis, early and late apoptosis and RIP1-dependent apoptosis phenotypes in live and dead cells. Necrostatin-1 and zVAD was shown to modulate levels of shikonin induced DDR, hyper-action of PARP and parthanatos in the four forms of RCD processes analysed. LC3B was up-regulated by combined treatment of zVAD with chloroquine which also revealed that DNA damage was reduced in live cells but enhanced in dead cells indicating the role of autophagy in maintaining cell health. This approach to RCD research should be a great advance to understanding the mechanisms of drugs and their effects upon RCD populations.

A Flow Cytometric Study of ER Stress and Autophagy.

The mechanistic link between ER stress, autophagy, and resultant cell death was investigated by the use of drugs Thapsigargin (Tg) and Chloroquine (CQ) with prior induction and or blockade of autophagy and apoptosis which modulated the ER stress response and resultant form of cell death. All these biological processes can be measured flow cytometrically allowing the determination of the type of cell death, G1 cell cycle arrest, cell cycle dependent measurement of ER stress transducer PERK, misfolded proteins, reticulophagy, and autophagy marker LC3B. Jurkat cells after Tg or CQ treatment became necrotic and apoptotic, showed G1 cell cycle arrest, autophagy, and ER stress. Prior induction of autophagy before ER stress increased levels of necrotic and apoptotic cell death. Autophagy was further up-regulated, while PERK was reduced or abrogated. CQ showed reduced levels of misfolded proteins and reticulophagy, while Tg showed no change in misfolded protein levels but increased reticulophagy and thus displayed more ER stress. Prior blockade of apoptosis before induction of ER stress resulted in cell survival except with high Tg levels which induced necrosis. Autophagy was up-regulated with modulation of PERK and reticulophagy levels with an abrogation of the misfolded protein response. Blockade of apoptosis with induction of autophagy before ER stress showed death by necrosis with high dose drugs and cell survival with low doses of drugs. CQ induced reduced levels G1 cell cycle arrest while it was maintained with Tg. Autophagy was also maintained with reduced levels of ER stress. These data demonstrates a profound link between the processes of ER stress, autophagy, and the resultant form of cell death all of which can be modulated depending upon the sequence and concentration of drugs employed. © 2018 International Society for Advancement of Cytometry.

Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis.

Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population. Here we describe a flow cytometric assay fully realising that potential by the use of anti-RIP-3 (Receptor-interacting serine/threonine-protein kinase 3) and anti-active caspase-3 fluorescently tagged antibodies and a fixable live dead fluorescent dye. This allows the determination of the degree of necroptosis, apoptosis and RIP1-dependent apoptosis within live and dead populations. Necroptosis was identified by the up-regulation of RIP3, while RIP1-dependent apoptosis was described by double positive for RIP3/active Caspase-3 events in live and dead populations. Apoptotic cells were defined by an active-Caspase-3+ve/RIP3-ve phenotype. Pan-caspase blocker zVAD and RIP1 inhibitors GSK'481 or necrostatin-1 revealed interesting modulations of such sub-populations of Jurkat cells. This novel flow cytometric assay employing two antibodies and a fixable viability probe provides the researcher with in-depth analysis of various forms of regulated forms of cell death beyond what is currently available and is a major methodological advancement in this field.

Flow cytometric assays for the study of autophagy.

The use of flow cytometry to study the autophagic process has recently led to the development of numerous assays measuring various aspects of the autophagic process. These include the detection of the autophagy marker, the microtubule associated protein LC3B, cell cycle analysis of LC3B expression, increase in lysosomal mass, as well as organelle specific autophagy and the measurement of mitochondrial function. We employed a range of autophagy inducing agents to determine the degree of LC3B up-regulation and corresponding cell cycle distribution, increase in lysosomal mass and mitochondrial dysfunction, as well as the relative preference for the specific type of microautophagy or organelle phagy. A variety of autophagy inducing agents were compared these included rapamycin, chloroquine, various nutrient starvation treatments on two cell types, Jurkat T-cell leukaemia and K562 erythromyeloid leukaemia cell lines. Given that numerous autophagy inducing agents cause cell cycle arrest, the cell cycle phase distribution was investigated and LC3B antigen was shown to increase as cells progressed through the cell cycle. LysoTracker dyes have been previously employed to investigate the autophagic process and here the LysoTracker signal increased in autophagic cells in relation to controls. Organelle autophagy of mitochondria and Endoplasmic Reticulum (ER), termed mitophagy and ER phagy was determined flow cytometrically by the employment of organelle mass probes, MitoTracker Green (MTG) and ER Tracker Green (ERTG). A modification of the cell cycle analysis width and area analysis employed for DNA content determinations was developed to show changes in organelle mass on a linear scale. Relative changes in linear scaled median fluorescence intensity (MFI) was compared to control cells to determine the degree and type of organelle phagy induced by a range of autophagy inducing agents and treatments. These flow cytometric organelle phagy and lysosome mass assays can be used by researchers to study the autophagic process further in terms of cell and mitochondrial functionality over time in a cell dependent manner as an adjunct to LC3B measurements.

Cell death pathways and autophagy in the central nervous system and its involvement in neurodegeneration, immunity and central nervous system infection: to die or not to die--that is the question.

Death rules our lives. In this short paper, we summarize new insights into molecular mechanisms of neurodegeneration. Here we review the most important processes of cell death: apoptosis and oncosis. We focus on autophagy, which is pivotal for neuronal homeostasis, in the context of neurodegeneration, infection and immunity. Its dysfunction has been linked to several neurodegenerative diseases such as Parkinson's, Huntington's and Alzheimer's diseases. Our understanding is still incomplete, but may highlight attractive new avenues for the development of treatment strategies to combat neurodegenerative diseases.

Real-time flow cytometry for the kinetic analysis of oncosis.

The standard method of distinguishing apoptotic and oncotic cells has been by microscopic analysis of nuclei and cell membrane morphology. Thus a rapid test for analyzing large numbers of cells in the study of cell necrobiology has not been possible until the recent advent of the Amnis Image-stream and real-time Lab-on-a-Chip technologies. An interesting difference between apoptosis and oncosis is that they are ATP dependent and independent processes, respectively. Here we describe an assay measuring real-time kinetic changes in the potential differences of the inner mitochondrial membrane (mmp) and the plasma membrane (pmp) in cells immediately before and after the addition of the inducing agent. Live cells were loaded with carbocyanine dye DiIC(1) (5) and bis-oxonol (DiBAC(4) (5)) to measure mmp and pmp in conjunction with annexin V-FITC and DAPI labeling for gating out annexin V binding cells and dead cells respectively. Live cells gave specific membrane signatures in response to apoptotic or oncotic reagents in real-time. Apoptosis showed little change in mmp and pmp signals over the course of 25 min, the mitochondria only showed a slight hyperpolarization. In contrast chemical treatment with oxidative phosphorylation blocker, sodium azide (SA) caused an immediate hyperpolarization spike followed by a complete abrogation of mmp over a 25 min time course. Treatment with SA (1%) also caused plasma membrane depolarization. Likewise detergent (0.01% Triton X-100) treatments also caused abrogation of mmp and depolarization of pmp. Whereas heat shock (42°C) treatment showed only a slight mitochondrial membrane potential depolarization. These flow cytometric observations were confirmed by confocal microscopy. This novel real-time kinetic assay measuring mitochondrial and plasma membrane potential changes has important implications in the field of cell necrobiology in that it allows the researcher to differentiate apoptotic and oncotic processes in an immediate manner for the first time.

Quality management systems in ART: are they really needed? An Australian clinic's experience.

As assisted reproductive technology (ART) expanded globally, several countries introduced prescribed requirements for treatment and monitoring of outcomes, as well as a licensing or accreditation requirement. While it is common for ART laboratories to be required to have an effective quality control system, the remainder of the clinic is often under less stringent regulation. Furthermore, when treatment conditions are prescribed, the standards tend to be conservative and clinics may choose to establish their own standards. Total quality management systems are now being used by an increasing number of ART clinics. In Australia and New Zealand, it is now a requirement to have a quality management system in order to be accredited and to help meet customer demand for improved delivery of ART services in these two countries.

The effect of pentoxifylline on spontaneous and experimental metastasis of the mouse Neuro2a neuroblastoma.

Pentoxifylline (PTX) has been reported to have both direct and indirect anti-tumor effects in experimental tumor models. We studied the effect of PTX on (1) the proliferation of Neuro2a mouse neuroblastoma cells in vitro and in vivo, (2) spontaneous and experimental metastasis, (3) tumor cell membrane fluidity and (4) adhesion to a fibronectin-coated surface. PTX significantly reduced the proliferation of Neuro2a cells in vitro as determined by DNA measurement (P < 0.01) and total cell count (P < 0.02). In vivo, PTX reduced the growth of subcutaneously transplanted primary tumors in syngeneic A/J mice (P < 0.01; n = 15). All seven animals (100%) receiving intravenous tumor cells developed extensive liver metastasis. In contrast, only 1/11 (9%) of animals pre-treated with oral PTX and injected with PTX-treated cells developed liver metastases. Of five mice receiving PTX-treated cells without oral pretreatment of PTX, two out of five (40%) developed liver metastases. There was a slight, but not significant (P = 0.08) increase in both experimental and spontaneous lung metastases formation in PTX-treated animals. However, tumor nodule formation on the lung surface was inefficient. PTX also increased membrane fluidity of the Neuro2a cells and significantly decreased tumor cell adhesion to fibronectin-coated microtiter wells (P < 0.01). We conclude that PTX has a cytostatic effect on the Neuro2a mouse neuroblastoma and exerts an anti-tumor effect on liver metastases following intravenous administration of neuroblastoma cells. Whether these results are directly related to the changes in membrane properties caused by pentoxifylline remains to be established.

The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor.

Tissue factor (TF), the membrane glycoprotein that initiates blood coagulation, is constitutively expressed by many tumor cells and is implicated in peri-tumor fibrin deposition and hypercoagulability in cancer. Upregulation of tumor TF correlates with enhanced metastatic potential. Furthermore, TF has been colocalized with VEGF in breast cancer, specially at sites of early angiogenesis. There are no data on the effect of hypoxia on tumor cell TF expression. Since hypoxia is known to stimulate VEGF production, we studied whether this also induces tumor cell TF expression. Confluent monolayers of A375 melanoma, MCF-7 breast carcinoma and A549 lung carcinoma were cultured in either 95% air, 5% CO2 (normoxic) or 95% N2, 5% CO2 (hypoxic; 25-30 mmHg) for 24 h. Procoagulant activity (PCA) was measured by amidolytic and clotting assays, surface TF antigen by flow cytometry, early apoptosis by annexin V binding and VEGF levels in culture supernatants by ELISA. Hypoxia significantly increased tumor cell PCA in all three cell lines tested and TF antigen on A375 cells was increased four-fold (P <0.05). Pentoxifylline (PTX), a methylxanthine derivative, significantly inhibited the hypoxia-induced increase in PCA as well as VEGF release in all three cell lines tested. In A375 cells, PTX significantly inhibited TF antigen expression by both normoxic and hypoxic cells. Hypoxia induced a slight (5%) but not significant, increase in early apoptosis. Intravenous injection of hypoxic A375 cells into nude rats produced more pronounced thrombocytopenia (n = 5, P <0.01) and more lung metastases (n = 3, P <0.05) compared to normoxic cells. We conclude that hypoxia increases TF expression by malignant cells which enhances tumor cell-platelet binding and hematogenous metastasis. Hypoxia-induced upregulation of TF appears to parallel that of VEGF, although the mechanism remains unclear.

Culture factors affecting the success rate of in vitro fertilization and embryo transfer.

The development of one-cell mouse zygotes to the blastocyst stage in vitro has been used as a quality control for the media and handling procedures employed for human in vitro fertilization and embryo transfer (IVF/ET). One-cell mouse zygotes were placed in culture in medium containing bovine serum albumin. Aliquots of the same batch of medium containing female patients' homologous serum were used for the fertilization and culture of human oocytes. The following procedures were associated with high rates of mouse embryo development and human pregnancies following IVF/ET: adequate gassing and equilibration of the medium, double-rinsing of pipets and catheters used to handle embryos, use of a HEPES-buffered medium for manipulating embryos in the absence of an atmosphere containing 5% CO2, control of excessive temperature in the vicinity of the embryos, and ET using medium containing 50% patient's serum. The institution of these procedures gave more consistent pregnancy rates. However, there was no obvious association between fertilization and cleavage of human oocytes and the quality of the medium ascertained by the mouse embryo development test. In a continuing trial, we are comparing two culture media (modified Tyrode's and a medium formulated on the composition of human fallopian tube fluid [HTF]) and two culture techniques (culture in medium under oil in petri dishes and in loosely capped tubes). Significantly more mouse zygotes developed in HTF medium compared to Tyrode's medium. In a randomized 2 X 2 factorial trial with human IVF/ET, the highest pregnancy rate occurred when fertilization and culture were carried out in HTF medium under oil, but numbers are not yet sufficient to show any statistical difference between treatments.

The effect of transferring pronuclear embryos on pregnancy outcome after in vitro fertilization.

In a prospective clinical trial the pregnancy rate in patients matched for infertility status, degree of hyperstimulation, and number of oocytes recovered was unaffected by whether embryos were transferred while still pronuclear (day 1) or after they had undergone cleavage (day 2). The pregnancy rates per transfer were 27% and 22%, respectively, for the two transfer times. Unlike results of a previous study, no difference was detected in the outcome of pregnancies from the two groups.

The application of flow cytometry to the study of bacterial responses to antibiotics.

Experiments were performed to determine whether a modern flow cytometer could be used to study bacterial populations in suspension, with particular reference to their morphological characteristics and their responses to antibiotics. The FACScan, a commercial benchtop flow cytometer fitted with an air-cooled laser, designed primarily for the study of eukaryotic peripheral blood mononuclear cells, yielded reproducible data relating to bacterial shape and internal architecture. It was sensitive enough to detect changes in bacterial morphology on entry into the growth cycle and after exposure to antibiotics. Antibiotic-induced morphological changes affecting subpopulations of bacteria were sufficiently specific to allow differentiation between antibiotics with different cell-wall enzyme targets. Simultaneously, the effect of such antibiotics on the integrity of the outer cell membrane of Escherichia coli was assessed by measurement of the association of the nucleic acid-binding dye propidium iodide with the bacteria. These experiments demonstrated complex patterns of probable cell-wall leakage, related to the modes of action of the antibiotics. The FACScan is a useful and sensitive tool for the study of the morphology and physiology of bacterial populations in suspension, and is especially applicable to the study of antibiotic action.

Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid.

Significantly more mouse zygotes developed to blastocysts in culture in a medium formulated on the composition of human tubal fluid (HTF) than in modified Tyrode's medium (T6). In a randomized 2 X 2 factorial trial of human in vitro fertilization that compared the two media and culture under oil versus culture in loosely capped tubes, significantly more clinical pregnancies (30% of 60 transfers) were obtained with HTF medium than with T6 medium (11% of 53 transfers). Decreasing the K+ content of HTF medium to that present in T6 medium significantly decreased the number of mouse zygotes that developed in culture. Modifying Ca++ levels had no effect. It is therefore likely that the higher K+ content in HTF medium is primarily responsible for the superiority of HTF medium over T6 medium, but other differences in the composition of the two media could contribute to the results observed.

Pregnancy-related chemotactic activity of human follicular fluid.

We measured chemotactic activity in 238 follicular fluids (FF) aspirated from 45 women who had undergone ovarian stimulation with a combination of clomiphene citrate and human menopausal gonadotropin for oocyte retrieval, in vitro fertilization, and embryo transfer. Fifteen of the treatment cycles resulted in pregnancy. The mean chemotactic activity, measured as the distance in microns granulocytic leukocytes migrated through a 3.0-micron membrane, was significantly higher in FF from conceptual cycles, compared with nonconceptual cycles. Serum chemotactic activity was significantly lower in conceptual cycles, compared with nonconceptual cycles. A chemotactic gradient appears to exist between the peripheral circulation and the ovarian follicle. The gradient favors the follicle in conceptual cycles, as indicated by the chemotactic quotient (the ratio of chemotactic activity of FF to serum). In conceptual cycles the chemotactic quotient was 1.7 +/- 0.17, compared with 0.7 +/- 0.03 for nonconceptual cycles. The presence of leukocyte chemotactic factor in FF appears to discriminate prospectively with a 90% degree of confidence between conceptual and nonconceptual in vitro fertilization and embryo transfer cycles.

Culture factors in relation to the success of human in vitro fertilization and embryo transfer.

The development of 1-cell mouse zygotes to the blastocyst stage in vitro has been used as a quality control for the media employed for human in vitro fertilization and embryo transfer (IVF-ET). The following procedures were associated with high rates of mouse embryo development and human pregnancies following IVF-ET: adequate gassing and equilibration of the medium, double rinsing of pipettes and catheters used to handle embryos, use of a HEPES-buffered medium for manipulating embryos in the absence of an atmosphere containing 5% CO2, control of excessive temperature in the vicinity of the embryos, and ET using medium containing 50% patient's serum. The institution of these procedures gave more consistent pregnancy rates. However, there was no obvious association between fertilization and cleavage of human oocytes and the quality of the medium ascertained by the mouse embryo development test.

Failure of body mass index or body weight to influence markedly the response to ovarian hyperstimulation in normal cycling women.

A retrospective analysis was performed of 368 normally cycling women treated with a single cycle of a standard ovarian hyperstimulation regime (CC 100 mg days 5 to 9 and hMG 150 IU days 6, 8, and 10) associated with either an IVF or GIFT program. Neither the peak serum E2 level attained nor the number of days of stimulation required bore a relationship to the BMI or the total body weight of these women. Whereas the mean number of oocytes aspirated from women with BMI less than 19.1 was higher (6.4 +/- 3.2) compared with obese women (BMI greater than 27.6, 4.8 +/- 2.6), the rate of fertilization was not different for both BMI extremes. It is concluded that factors other than BMI or total body weight have more important influences on the response to hyperstimulation in normal women.

A prospective trial of intrauterine insemination of motile spermatozoa versus timed intercourse.

STUDY OBJECTIVE: The efficacy of intrauterine insemination (IUI) of selected motile sperm. DESIGN: Prospective randomized sequential alternating cycle trial comparing IUI with luteinizing hormone (LH)-timed intercourse. SETTING: Clinical infertility service. PATIENTS: Couples selected included unexplained infertility (n = 73), cervical mucus hostility (n = 24), moderate semen defect (n = 110), and severe semen defect (n = 78). Two hundred eighty-five couples undertook 600 IUI cycles and 505 LH-timed intercourse. RESULTS: Overall, IUI was slightly more effective than LH-timed intercourse with a pregnancy rate of 6.2% versus 3.4% per cycle. When individual categories were considered only, IUI for severe semen defect was significantly better (5.6% versus 1.3%, P less than 0.05). The first IUI cycle was more effective when compared with both subsequent IUI cycles and the initial LH-timed cycle. Overall, 74% (27/37) of IUI pregnancies occurred in the first cycle. CONCLUSIONS: Compared with LH-timed intercourse, IUI provided little or no improved expectation of pregnancy but was beneficial in couples with severe semen defect. The occurrence of pregnancy was limited per cycle and confined essentially to the initial cycle of treatment. Continued IUI is considered to be unrewarding.

Overweight infertile patients have a higher fecundity than normal-weight women undergoing controlled ovarian hyperstimulation with intrauterine insemination.

There is a positive effect of being overweight or obese on the pregnancy rate in women undergoing controlled ovarian hyperstimulation with intrauterine insemination.