Apoptosis and necrosis are the two major forms of cell death mechanisms. Both forms of cell death are involved in several physiological and pathological conditions and also in the elimination of cancer cells following successful chemotherapy. Large number of cellular and biochemical assays have evolved to determine apoptosis or necrosis for qualitative and quantitative purposes. A closer analysis of the assays and their performance reveal the difficulty in using any of these methods as a confirmatory approach, owing to the secondary induction of necrosis in apoptotic cells. This highlights the essential requirement of an approach with a real-time analysis capability for discriminating the two forms of cell death. This paper describes a sensitive live cell-based method for distinguishing apoptosis and necrosis at single-cell level. The method uses cancer cells stably expressing genetically encoded FRET-based active caspase detection probe and DsRed fluorescent protein targeted to mitochondria. Caspase activation is visualized by loss of FRET upon cleavage of the FRET probe, while retention of mitochondrial fluorescence and loss of FRET probe before its cleavage confirms necrosis. The absence of cleavage as well as the retention of mitochondrial fluorescence indicates live cells. The method described here forms an extremely sensitive tool to visualize and quantify apoptosis and necrosis, which is adaptable for diverse microscopic, flow cytometric techniques and high-throughput imaging platforms with potential application in diverse areas of cell biology and oncology drug screening.

Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays.Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application.Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe.Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology.

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Recent cell biology studies reveal that a cell can die through multiple pathways via distinct signaling mechanisms. Among these, apoptosis and necrosis are two distinct cell death pathways, and their detection and discrimination is vital in the drug discovery process and in understanding diverse biological processes. Although sensitive assays for apoptosis and necrosis are available, it is extremely difficult to adapt any of these methods to discriminate apoptosis-inducing stimuli from necrosis-inducing stimuli because of the acquisition of secondary necrosis by apoptotic cells when they are not phagocytosed. Essentially, any assay for discriminating apoptosis and necrosis needs to be carried out in real-time kinetic mode. Caspase 3 or 7 activation is observed in the majority of apoptotic cell death. Similarly, the absence of caspase 3/7 activation and cell membrane leakage are the two prominent indicators for necrotic cell death or necroptosis. The programmed form of necrosis, called pyroptosis, is also accompanied by membrane leakage and most often associated with activation of specific caspases such as caspase 1, 4, or 11, but not through caspase 3/7 activation. Here, a robust and sensitive real-time method is described to distinguish and discriminate apoptosis from necrosis. The assay utilizes stable integration of a genetically encoded fluorescence resonance energy transfer (FRET) probe for caspase 3/7 activation and the mitochondrion-targeted DsRed to identify necrotic cells. Caspase activation is determined by cleavage of the FRET probe; loss of soluble FRET probe with retention of mitochondrial red fluorescence indicates necrosis. This unit describes an important protocol for the generation of sensor cells expressing both probes, followed by detailed analysis of apoptosis and necrosis by microscopy imaging, confocal imaging, high-throughput imaging, and flow cytometry. © 2018 by John Wiley & Sons, Inc.

Cancer cells grown as 3D‐structures are better models for mimicking in vivo conditions than the 2D‐culture systems employable in drug discovery applications. Cell cycle and cell death are important determinants for preclinical drug screening and tumor growth studies in laboratory conditions. Though several 3D‐models and live‐cell compatible approaches are available, a method for simultaneous real‐time detection of cell cycle and cell death is required. Here we demonstrate a high‐throughput adaptable method using genetically encoded fluorescent probes for the real‐time quantitative detection of cell death and cell cycle. The cell‐cycle indicator cdt1‐Kusabira orange (KO) is stably integrated into cancer cells and further transfected with the Fluorescence Resonance Energy Transfer‐based ECFP‐DEVD‐EYFP caspase activation sensor. The nuclear cdt1‐KO expression serves as the readout for cell‐cycle, and caspase activation is visualized by ECFP/EYFP ratiometric imaging. The image‐based platform allowed imaging of growing spheres for prolonged periods in 3D‐culture with excellent single‐cell resolution through confocal microscopy. High‐throughput screening (HTS) adaptation was achieved by targeting the caspase‐sensor at the nucleus, which enabled the quantitation of cell death in 3D‐models. The HTS using limited compound libraries, identified two lead compounds that induced caspase‐activation both in 2D and 3D‐cultures. This is the first report of an approach for noninvasive stain‐free quantitative imaging of cell death and cell cycle with potential drug discovery applications.

Background Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. Methods We describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies/metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2-Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. Results The first approach demonstrates single-color fluorescent imaging of ACE2 –GFPSpark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening. Life-time imaging of the ACE2-GFPSpark confirmed the interaction of ACE2 and S1-OFPSpark on beads. Conclusions The self-renewable source of secreted recombinant proteins from stable cells and its direct use without necessitating purification renders the platform a cost-effective and rapid one than the popular pseudovirion assay and live virus-based assays. Any laboratory with minimum expertise can rapidly perform this bead assay for neutralizing antibody detection using stable engineered cells. Statement of significance The bead-based assay platform for screening neutralizing antibodies against SARS CoV-2 is a cost-effective alternative for the gold standard pseudovirion assay. This assay will accelerate our efforts to develop newer vaccines against COVID-19 and can be used to discover viral entry blockers engaging S1-ACE2 in drug screening settings.