Notes

Tried altruistic infanticide in the circumstance regarding psychotic decompensation induced simply by stress linked to the COVID-19pandemic: a case document.
Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to capture the kinetic profiles of such responses in a dynamic and noninvasive way can further advance the utility of 3D cell cultures. Here, we describe the use of a luminescent lactate dehydrogenase (LDH) toxicity assay for monitoring time- and dose-dependent effects of drug treatment in 3D cancer spheroids. HCT116 spheroids formed in 96-well ultralow attachment plates were treated with increasing drug concentrations. Thymidine Medium samples were collected at different timepoints, frozen, stored, and analyzed at the end of experiments using the luminescent LDH-Glo™ Assay. High assay sensitivity and low volume sampling enabled drug-induced toxicity profiling in a time- and dose-dependent manner.Anoikis is a type of programmed cell death triggered by the loss of cellular interaction with the extracellular matrix (ECM) and culminates in the activation of caspases. Specific interaction between cellular receptors such as integrins and the ECM is important to maintain cellular homeostasis in normal tissues through multiple cascades. This interaction provides not only physical attachment, but more importantly, vital interaction with the actin cytoskeleton and growth factors. Normal epithelial and endothelial cells require this interaction with ECM to survive. In cancer, the acquisition of anoikis resistance is a hallmark of malignant transformation and is required in the process of metastasis formation. As such, strategies to inhibit and/or counteract anoikis resistance are important in controlling cancer progression. In this chapter, we describe the method for detecting anoikis using cell viability and caspase activity assays.This chapter describes a real-time, bioluminescent apoptosis assay technique, which circumvents the well-documented "timing condundrum" encountered when employing traditional apoptosis detection chemistries after exposures with inducers of unknown potential. The assay continuously reports the translocation of phosphatidylserine (PS) from the inner membrane leaflet of a cell to the exofacial surface during apoptosis. This homogenous, no-wash, plate-based assay is made possible by two different annexin V fusion proteins, which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During apoptosis, luminescence signal is proportional to PS exposure and fluorescence intensity correlated with the degree of secondary necrosis. Altogether, the measures provide exquisite kinetic resolution of dose- and agent-dependent apoptotic responses, from early through late phases. At exposure termination, other compatible reagents can be applied to measure additional orthogonal correlates of cell health.Phenotypic analysis of the effects of a gene of interest may be limited because stable expression of some genes leads to adverse consequences in cell survival, such as disturbance of cell cycle progression, senescence, autophagy, and programmed cell death. One of the best examples is tumor suppressor p53. p53 functions as a tumor suppressor by inducing cell cycle arrest and apoptosis in response to genotoxic and environmental insults. The choice and timing of either pathways induced by p53 depend on cellular context, cell types, and the degree of cellular/genomic damage (For review, see (Chen J, Cold Spring Harb Perspect Med 6a026104, 2016)). The uncertainty makes the studies on the long-term effects of p53 in cells challenging. This chapter describes a method of flow cytometric analysis of ectopic expression of p53 to better quantify cell cycle distribution and apoptosis in cells treated with DNA damaging agents. The method can be easily adapted to other genes of interest to study their contributions to the fate of variety of cell types in response to endogenous or exogenous stresses.Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.
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