Reveal Cellular Secrets With Live-Cell Imaging

Unlocking cellular secrets with live-cell imaging. Live-cell imaging solutions 2 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Introduction Cellular imaging is a well-established approach to the study of cellular functions, behaviors, and pathways to gain a deeper understanding of disease mechanisms and treatment responses. Unlike traditional fixed-endpoint cell assays, which provide only a point-in-time snapshot of cellular responses, live-cell imaging delivers a fuller, more realistic picture of the effects of perturbations. Researchers can observe when and how fast something happened, i.e., the kinetics of a cellular reaction, which helps them better understand cellular responses. Application Typical Duration Ca2+ flux Milliseconds Cell migration Minutes Protein translocation Minutes Plasmid transfection Approx. 24h Scratch wound Approx. 24h Cell health & viability 1 day to a few days Immune-cell killing 1 day to a few days Spheroid health Several days Stem cell monitoring Days to weeks Live-cell imaging assays can be categorized as follows: Diverse assays, dynamic timescales Live-cell end-point assay A single image of live cells captured after a specified incubation period. Kinetic live-cell assays Multiple sequential images acquired over a time course of hours, days, or weeks. Fast response assays Images captured immediately after dispensing a compound or modulator into wells. The timing of image acquisition is dependent on the known or expected response speed, which may be milliseconds. Diversity of live-cell assays. Dynamic cellular events such as proliferation, migration, movement, signaling, apoptosis, cytotoxicity, and morphological changes can all be studied by imaging live cells. These dynamics occur over a broad range of timescales. 3 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Benefits of live-cell imaging Real-time to long-term Cell migration: single cell tracking Migration of live cells can be studied using high-content imaging. Brightfield or digital phase contrast imaging using a red LED minimizes phototoxicity. Software tools enable tracking of individual cells, providing kinetic readouts such as the speed and distance traveled. Additionally, morphological profiles can be generated and correlated with migration behavior, offering more detailed insights into cellular movement patterns. Immune-cell killing assays Immune-cell killing assays represent a key approach in the study of immunooncology— the harnessing of the immune system to combat cancer. High-content analysis of T-cell mediated killing of cancer cells in real-time enables the detailed study of potential immunotherapies, providing insights into both the kinetics and mechanisms of immune responses. Calcium flux assays Measuring the rapid influx of calcium into the cell cytosol is important in the study of intracellular signaling and pathological conditions such as impaired synaptic transmission or muscle contraction. The very fast (milliseconds) movement is detected in live cells using a calciumsensitive dye and an imaging system with “dispense and read” capability. MILLISECONDS / SECONDS MINUTES HOURS 4 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Benefits of live-cell imaging Six benefits of live-cell imaging Visualization of dynamic processes and cellular events in real-time y Cell migration and motility (e.g., wound healing, chemotaxis) y Organelle trafficking and dynamics (e.g., mitochondria movement, endocytosis) y Cell division and mitosis (e.g., tracking mitotic phases) y Protein translocation and signaling events (e.g., nuclear translocation of transcription factors) 1 Study of long-term processes y Differentiation, apoptosis, or autophagy progression over hours/days y Interactions between live cells, such as immune cell activity or synapse formation 4 Avoiding fixation-induced artifacts y Depending on the type of study, it may be beneficial to avoid fixation reagents as they could influence the sample in an uncontrolled way or alter protein localization y Especially critical when studying protein trafficking, where fixation might lead to artificial redistribution of proteins of interest 5 Simplification of experimental workflow y Live-cell assays are often “mix and run”, whereas immunofluorescence staining requires multiple washing steps between antibody incubations 6 Observation of cellular responses to treatments over time y Tracking responses to drugs or environmental changes dynamically y Monitoring transient signaling events that may be lost during fixing y Studying cell recovery from stress (e.g., phototoxicity, osmotic shock) 2 Maintenance of cell viability and physiology y Preserves physiological conditions (e.g., pH fluctuations, ion gradients) 3 5 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Overcoming the challenges Navigating the challenges of live-cell imaging Maintaining cell health Efficiency Imaging Environmental conditions Data handling Dyes and stains Phototoxicity Scalability & throughput Image Quantification/ Analysis/Segmentation 6 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Overcoming the challenges Maintaining cell health Revvity solutions: Maintaining cell viability and normal function throughout the duration of experiments, which may extend to several weeks, presents a major challenge in live-cell imaging. Factors impacting cell health include: y Environmental conditions: Temperature, CO2 concentration, and humidity should be controlled over the entire time course with minimal disruption. y Light exposure: Prolonged exposure to excitation light can be toxic to live cells, potentially altering cellular processes. y Imaging reagents: Dyes may be toxic to the cells over prolonged timescales or become toxic on excitation (phototoxicity), interfering with normal cellular processes and behavior. Environmental control y Fluctuation-resistant environmental control unit Reduced light exposure y Automated water immersion lenses are more sensitive and allow shortening of exposure times y Confocal image acquisition removes much of the light from outside the focal plane. Label-free high-content analysis y Analyzes brightfield images, avoiding the use of a fluorescent nuclear stain. y Identifies the cytoplasm in AI-generated digital phase contrast (DPC) images. Label-free imaging y Rapid whole well label-free analysis for cell-based assays Non-toxic dyes y Specifically designed for live-cell staining y Even in experiments lasting more than 24 hours, cytotoxicity can be mitigated by using appropriate concentrations Opera Phenix™ Plus & Operetta CLS™ high-content imaging systems Phenologic.AI™ software Celigo™ image cytometer PhenoVue™ cellular imaging reagents 7 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Overcoming the challenges Imaging and analysis of live cells Revvity solutions: Staining y Stains and dyes may fade over the time-course of the assay or as cells divide (photo-bleaching) y Background signal from culture medium may make a dye more difficult to detect than in a fixed-cell assay Image quantification y Brightfield images, which may be preferred for long-term experiments or cell-culture applications, are difficult to quantify due to low contrast y Classical image analysis algorithms are optimized for fluorescent images Analysis of Brightfield and DPC images y Simplifies the detection of cellular nuclei in brightfield images y Employs AI to calculate digital phase contrast (DPC) images for precise cytoplasm identification. y Works effectively with living cells, allowing segmentation, counting and even tracking of cells without dyes over time. Brightfield imaging y Flat illumination and excellent edge-to-edge contrast allows for enhanced image quality. Bright dyes y Specifically designed for live-cell staining y Maintain brightness and high image quality in experiments lasting more than 24 hours Phenologic.AI software Celigo image cytometer PhenoVue cellular imaging reagents 8 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Overcoming the challenges Efficiency of live-cell assays Revvity solutions: Throughput limitations y Live cell assay throughput is limited by the requirement to image the same plate multiple times. y Rapid cellular processes (e.g., cell cycle phases) may be missed between measurement cycles because the time required for a complete plate scan is too long. y Maintaining cell health during transfer and imaging of multiple plates presents significant challenges. Data handling y Live-cell imaging with multiple time points produces a large amount of data, creating an image analysis bottleneck and storage challenges. Fast image acquisition y Multiple cameras for simultaneous imaging, significantly reducing imaging time and increasing throughput. Fast image analysis y >5x faster data transfer than previous versions. y Simultaneous image acquisition and automated data transfer, streamlining workflow efficiencies. Fast image acquisition y Routine cell-based assays, counting and imaging of individual cells in each well at high speed. Advanced data management y Provides very fast image analysis and affordable data storage. Automation y Enables significantly higher throughput and greater efficiency, allowing analysis of more cells across more plates over longer time periods. Opera Phenix Plus high-content imaging system Harmony™ high-content imaging & analysis software Celigo image cytometer Signals Image Artist software Revvity robotic platforms 9 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Applications of live-cell imaging Kinetic analysis of calcium flux activity in human iPSC-derived neurons using the Opera Phenix Plus system. In this study PhenoVue Cal-520 AM calcium indicator was used with fast kinetic imaging (5 fps) to monitor spontaneous calcium signaling in single neurons. Pharmacological interventions using Glutamate (excitatory neurotransmitter) and Gabazine (GABA receptor antagonist) were tested. Results showed Glutamate increased calcium response activity 9-fold in cell bodies and 5.5-fold in neurites, while Gabazine inhibited activity up to 4-fold. The automated liquid handling system of the Opera Phenix Plus enabled real-time compound addition during imaging. This approach provides reliable quantification of neuronal calcium dynamics, offering valuable insights for neuroscience research and potential drug screening applications in neurodegenerative disease studies. Fast kinetic calcium flux imaging using the Opera Phenix Plus highcontent screening system. This study demonstrates fast kinetic calcium flux imaging for GPCR analysis using CHO-K1 cells expressing histamine H1 receptors. Cells were loaded with Cal-520 calcium indicator and imaged at 68 fps following automated compound addition. For agonist testing, histamine showed dose-dependent calcium release with an EC50 of 22 nM. In antagonist assays, pyrilamine pre-treatment inhibited histamine-induced calcium flux with an EC50 of 117 nM. Single-cell analysis enabled identification of subpopulations including low/high responders and false positives. The synchronized liquid handling and high-speed imaging (up to 100 fps) captured rapid calcium responses occurring within milliseconds, providing robust quantification for GPCR drug screening applications. Kinetic analysis of calcium flux activity in human iPSC-derived neurons using the Opera Phenix Plus system A P P L I CAT I O N N OT E Abstract The study of intracellular dynamic processes is of fundamental importance for unraveling the mechanisms of a broad spectrum of diseases and to develop effective drugs and therapies. Particularly in the field of neuroscience and neurodegenerative diseases calcium signaling is of critical importance. Calcium ions act as second messenger molecules that elicit responses such as neurotransmitter release from synaptic vesicles and altered gene expression. The calcium concentration is highly dynamic due to the presence of intracellular calcium stores and pumps that selectively transport these ions in response to a variety of signals. Here we apply fast kinetic imaging using the Opera Phenix Plus high-content screening system to visualize and evaluate spontaneous calcium flux activity in single human iPSC-derived neurons labeled with PhenoVue™ Cal-520 AM. Our approach enables the assessment of calcium flux activity in single neurons, crucial for understanding the fundamental processes of neuronal communication and potential disruptions associated with neurodegenerative disease or neurotoxic insults. We used Gabazine as an antagonist of GABA(A) receptors and Glutamate as an abundant excitatory neurotransmitter to demonstrate the sensitivity of the assay to pharmacological interventions.1 Key features • Fast frame rate imaging to accurately capture rapid cellular responses • Reliable quantification of calcium activity in single neurons • Measure immediate compound responses using onboard liquid handling Revvity application notes for live-cell imaging 10 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Applications of live-cell imaging Revvity application notes for live-cell imaging Label-free analysis of cardiomyocyte beating using the Opera Phenix Plus system. This application note demonstrates label-free analysis of cardiomyocyte beating using the Opera Phenix Plus system with fast kinetic imaging up to 100 fps. Human iPSC-derived cardiomyocytes were cultured and monitored for contractility changes in response to cardiotoxic compounds. Brightfield imaging proved non-phototoxic while maintaining stable environmental conditions. Testing revealed epinephrine increased beating frequency by 35% at 1 μM, ivabradine caused dose-dependent decreases, and nifedipine completely eliminated beating at 0.005 μM. The automated liquid handling enabled real-time compound addition during imaging. This approach provides reliable, scalable cardiotoxicity screening for drug development, offering precise quantification of compound-induced changes in cardiomyocyte contractility under physiological conditions. Analysis of mitochondrial dynamics in human iPSC-derived neurons using the Operetta CLS high-content analysis system. This study demonstrates how the Operetta CLS system was used to analyze mitochondrial dynamics in human iPSC-derived neurons through fast kinetic imaging at 2 fps. Neurons were labeled with PhenoVue 551 mitochondrial stain and treated with FCCP (mitochondrial uncoupler) and glutamate (excitatory neurotransmitter). The analysis quantified dynamic mitochondrial fraction by subtracting adjacent time points to identify moving mitochondria. Results showed FCCP significantly decreased mitochondrial movement in a dose-dependent manner, while glutamate showed no significant effect. The gentle imaging conditions preserved mitochondrial function for up to one hour, enabling reliable quantification of mitochondrial dynamics crucial for understanding neurodegeneration and developing therapeutic interventions. A P P L I CAT I O N N OT E Introduction Drug-induced cardiotoxicity is a frequent cause of drug attrition during drug development and can lead to the withdrawal of drugs from the market.1 In-vitro models using induced pluripotent stemcell derived cardiomyocytes are commonly used to predict both safety and therapeutic efficacy of drugs, chemicals, environmental pollutants, cosmetic ingredients, or food additives.2,3 The development of new, non-invasive approaches allowing the quick and reliable assessment of the cardiotoxic potential of a compound are necessary to improve drug safety.4 For research use only. Not for use in diagnostic procedures. Label-free analysis of cardiomyocyte beating using the Opera Phenix Plus system. Key features • Reliable quantification of cardiomyocyte beating frequency • Fast frame rate imaging up to 100 fps to accurately capture fast cellular responses • Tip-based on-board pipetting compatible with 96 and 384-well plates • Stable environmental conditions with temperature, CO2, and humidity control Figure 1: Opera Phenix Plus high content screening system. 11 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Applications of live-cell imaging Automated single cell tracking using the Operetta high-content analysis system: Analyzing chemokinesis of cancer cells. This study demonstrates automated single cell tracking using the Operetta system with digital phase contrast imaging to analyze cancer cell migration. A549 lung cancer cells were stimulated with EGF and tracked over time to measure chemokinesis. Optimization revealed 15-minute imaging intervals and 60-minute observation periods provided excellent Z’ factors above 0.5. EGF showed dose-dependent migration stimulation (EC50: 2.7 ng/mL), while AG-1478 inhibitor demonstrated dose-dependent suppression (IC50: 1.5 μM). Morphological profiling using 45 parameters identified distinct cell clusters correlating with migration speed, revealing that fastmigrating cells exhibit pronounced membrane ruffles. This approach enables robust quantification of cancer cell motility for drug screening applications. Automated single cell tracking using the Operetta high-content analysis system: Analyzing chemokinesis of cancer cells. Application A549 non-small cell lung cancer cells were cultured in F12-HAM (Sigma-Aldirch) supplied with 10% FCS and 2 mM L-Glutamine. To perform the assay, cells were seeded in serum-free medium at a density of 4500 cells per well into a 384-well CellCarrier™ microplate (Revvity, 6007558) freshly coated with 5 μg/cm² collagen I (BD Biosciences, 354236). After overnight cultivation, cell migration was stimulated by addition of EGF (Sigma-Aldrich, E9644) at different concentrations or by addition of 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, P1585). In the presence of 100 ng/mL EGF, the following inhibitory compounds were tested: AG-1478 (Cayman Chemicals, 10010244), Nocodazol (Sigma-Aldrich, M1404) and Cytochalasin D (Sigma-Aldrich, C8273). Live cell imaging was performed using an Operetta high-content analysis system equipped with a temperature and CO2 control option (TCO) set to 37 °C and 5% CO2. Directly after addition of the compounds, microplates were placed onto the pre-heated Operetta system and incubated for 30 min. After incubation, digital phase contrast images were acquired at 10X magnification (10X high NA objective) using Operetta’s automatic digital phase contrast algorithm. Phototoxicity is minimized because digital phase imaging uses a red light LED transmission light source. To analyze the morphology of migrating cells in detail, we acquired digital phase contrast images using a 20X high NA objective. Images were acquired for up to 6 h at imaging intervals of 5-15 min. Images were segmented using the Find Cells building block of the Harmony software, which provides a dedicated algorithm for segmenting digital phase contrast images. The segmented cells were subjected to cell tracking using the Track Objects building block. We calculated different properties that describe cell migration either on a per time point basis such as Current Speed or on a per cell track basis such as Displacement. Displacement is the direct line between the position of the first observation of a cell and the position of the last observation of a cell. This proved to be the most valuable readout for describing cell migration. These optimization studies were based on a migration experiment depicted in Figure 1, in which cells were stimulated to migrate by a treatment with 100 ng/mL EGF. The videos generated by the Harmony software showed an increased cell migration after stimulation with EGF, as indicated by the length of the cell tracks. Using this measurement, we tested how extending the interval between two consecutive images (imaging interval) in silico impacts on the primary readout value cell displacement. To this end, we considered either every time point for the analysis or only every second, every third, every fourth, and so on (Figure 2). We then plotted the cell displacement and the Z’ factor against the imaging interval. As panel 2B illustrates, extending the imaging interval beyond 15 min strongly impairs the Z’ factor due to a decrease in the cell displacement. This can be explained by cells moving too far between two images to be correctly tracked. Consequently, long tracks are split into two or more shorter tracks that have a much smaller displacement than the longer original cell track resulting in a decreased average cell displacement per well. Untreated 100 ng/mL EGF Revvity application notes for live-cell imaging Analyzing ERK signal transduction in live cells using a FRET-based ERK biosensor on the Operetta CLS high-content analysis system. This application note describes how a live cell FRET (Förster Resonance Energy Transfer) assay can be automated on the Operetta CLS highcontent analysis system to study ERK signaling. Using the EKAREV biosensor, the researchers monitored real-time modulation of the ERK pathway with activators and inhibitors, generating dose-response curves with excellent assay performance (Z’ value > 0.87). The combination of the optimized design of the EKAREV biosensor, high-quality imaging, and intuitive Harmony software enables reliable quantification of ERK activity. This assay supports drug discovery efforts targeting the Ras/Raf/MEK/ERK cascade by offering dynamic, highcontent analysis of living cells. A P P L I CAT I O N N OT E Introduction Extracellular signal-regulated kinase (ERK) is a key component in the regulation of embryogenesis, cell differentiation, cell proliferation and cell death.1 The ERK pathway originates from an activated receptor in the plasma membrane and is propagated via Ras/Raf/MEK to ERK (Figure 1). The pathway is activated by different types of receptors, including receptor tyrosine kinases (e.g. EGF receptor) as well as G-protein coupled receptors.2 As the final component of the signaling pathway, ERK phosphorylates different intracellular proteins including a large number of other kinases and transcription factors. The ERK signaling pathway is altered in various cancer types and hence is being investigated as a target for therapeutic intervention.3 Here, we describe how a live cell FRET (Förster Resonance Energy Transfer) assay, used to study ERK signaling, can be automated on the Operetta CLS™ high-content analysis system. This assay concept can support the drug discovery process for identifying signal-cascade modulating compounds. For research use only. Not for use in diagnostic procedures. Analyzing ERK signal transduction in live cells using a FRET-based ERK biosensor on the Operetta CLS high-content analysis system. Key Points: • Ratiometric high-content FRET assay in living cells • Quantify ERK signaling using an EKAREV biosensor • Study signaling cascademodulating compounds 12 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Applications of live-cell imaging Cytotoxicity studies on live primary human hepatocytes using the Operetta high-content analysis system To address the challenges of investigating drug-induced hepatotoxicity, researchers developed a live-cell cytotoxicity assay using primary human hepatocytes. Cryopreserved single-donor hepatocytes were treated with three model hepatotoxins and analyzed using a no-wash staining protocol. Live-cell imaging was performed on the Operetta high-content analysis system and five cellular parameters (cell count, roundness, nucleus texture, nuclear size, and mitochondrial texture) were quantified using Harmony software. This high content, multiparametric approach enables the assessment of hepatotoxic effects, supporting rapid toxicity profiling in a physiologically relevant liver model. was recorded on a standard Olympus® IX 50 microscope with a 10X objective (10X/0.30 Ph1). After 24 hr incubation with each compound and DMSO controls, a fluorophore dye cocktail containing Hoechst 33342, BOBO™-3 and MitoTracker® Deep Red (Invitrogen, H3570, M22426, B3586) for quantification of the nuclear size and staining pattern (texture). The use of MitoTracker® Deep Red enabled the detection of the cell shape and was also used to evaluate the cell roundness, as well as the distribution and quantity of Figure 1: Left. 10X phase contrast image of human primary hepatocytes seeded on collagen-coated 384-well CellCarrier™ microplates in a conventional monolayer, after thawing, overnight attachment and one wash step. The majority of the cells adhered and adopted a polygonal shape with a highly granulated cytoplasm; several binuclear cells could be observed. Most primary hepatocytes survived the thawing procedure, leaving few cells detached and rounded. Basal phenotype before drug treatment 100 μM Tacrine Control 3 μM FCCP 3 μM FCCP Revvity application notes for live-cell imaging 13 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Applications of live-cell imaging Live-cell assays with Revvity’s high-content imaging systems Viability analysis and high-content live-cell imaging for drug testing in prostate cancer xenograft-derived organoids. Van Hemelryk A, et al. Cells. 2023, 12(10), 1377 Van Hemelryk and colleagues created an optimized live-cell imaging workflow for drug testing in prostate cancer xenograftderived organoids using the Opera Phenix high-content screening system, enabling high-resolution visualization of organoids and quantification of cell death modalities to distinguish cytostatic from cytotoxic effects at the single-organoid level. High-throughput measure of mitochondrial superoxide levels as a marker of coronary artery disease to accelerate drug translation in patient-derived endothelial cells using Opera Phenix technology. Lee WE, et al. Int. J. Mol. Sci. 2024, 25(1), 22 Lee and colleagues used the Opera Phenix high-content screening system and Harmony software to measure mitochondrial superoxide (mROS), membrane potential, and mitochondrial area in patient-derived endothelial colony-forming cells, revealing elevated mROS in coronary artery disease cells versus healthy controls and identifying a receptor antagonist that reduced mROS. Pancreatic cancer organoids in the field of precision medicine: A review of literature and experience on drug sensitivity testing with multiple readouts and synergy scoring. Mäkinen L, et al. Cancers. 2022, 14(3), 525 Mäkinen and colleagues used the Opera Phenix system with Harmony software to study drug sensitivity in pancreatic cancer organoids, finding that some treatments reduced viability without causing cell death and tracking individual nuclei to reveal surviving cells after potent drug combinations, demonstrating the platform’s utility for uncovering mechanisms of resistance. REVVITY IN THE RESEARCH SPOTLIGHT 14 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Applications of live-cell imaging Live-cell assays with Revvity’s Celigo image cytometer MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis Ding L, et al. Cancer Medicine. 2024;13(13). Minichromosome maintenance protein family member 8 (MCM8) is an oncogene that predicts poor prognosis in gastric cancer (GC) patients. Knockdown experiments identified RPS15A as a downstream target of MCM8. Cell proliferation was evaluated using the Celigo image cytometer following MCM8 and RPS15A knockdown. High-throughput SARS-CoV-2 antiviral testing method using the Celigo Image Cytometer St Clair LA, et al. J Fluoresc 34, 561–570 (2024) A high-throughput screening method was developed using the Celigo image cytometer to rapidly evaluate drug candidate efficacy against SARS-CoV-2 infectivity. This approach also assessed drug cytotoxicity, providing a rapid alternative to plaque reduction assays for pandemic response. Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method McDonald J, et al. Cancer Research. 2024;84(6_Supplement):5930. A multiplexed apoptosis detection method was developed using the Celigo image cytometer to investigate the apoptotic effects of EP4A1, EP4A2, Paclitaxel, and Carboplatin on Kuramochi ovarian cancer cells. Multiplex staining enabled simultaneous assessment of early apoptosis, late apoptosis, and necrosis in a high-throughput format. Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method Chan LLY, et al. Cancer Research. 2023;83(7_Supplement):5326. The Celigo image cytometer was used to evaluate and compare two CAR T-cell-mediated cytotoxicity assays using GFP-expressing versus fluorescent dye-labeled myeloma and plasmacytoma cells. Analyses demonstrated that the GFP-based method provided higher sensitivity for detecting cytotoxicity levels compared to CMFDA/DAPI viability staining. REVVITY IN THE RESEARCH SPOTLIGHT 15 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Revvity solutions for live-cell imaging The PhenoVue live cell painting kit allows for multiplexed labeling of live cells using four fluorescent probes staining nuclei, mitochondria, microtubules, and intracellular vesicles. Designed for use in live-cell imaging workflows, the kit supports time-lapse acquisition to monitor phenotypic changes over time in response to chemical or genetic treatments. The probes are compatible with standard cell culture conditions and have been optimized to exert minimal impact on cell viability and proliferation, for reliable results even during extended imaging. By streamlining the staining workflow, the PhenoVue live cell painting kit reduces hands-on time and experimental variability to support the delivery of high-quality, reproducible data. Key features: y 4-Plex staining: Simultaneous visualization of nuclei, mitochondria, microtubules, and intracellular vesicles. y Time-lapse compatible: supports long-term imaging without perturbing cells. y Preserves proliferation and viability: Little impact on cell growth and health confirmed across multiple cell lines. y No-wash, straightforward protocol: Simple workflow that reduces handling and preserves live-cell conditions PhenoVue live-cell painting kit PhenoVue live-cell painting kit Time-lapse imaging of untreated U20S cells. Image acquisition every 8h for 72h on the Opera Phenix Plus high-content screening system. PhenoVue live-cell painting kits y Kit for 2 x 96-well plates y Kit for 10 x 96-well plates 16 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Revvity solutions for live-cell imaging Non-toxic PhenoVue live-cell stains are designed for detection of cell organelles, such as lysosomes, actin, and tubulin, in live cell assays. They offer bright and stable fluorescence (no photobleaching), a no-wash assay protocol, and have no observed effect on cell health or proliferation. PhenoVue live-cell stains PhenoVue Fluor 647 live-cell actin stain Time-lapse imaging of untreated U20S cells. Image acquisition every 15 min for 15h on the Opera Phenix Plus system. PhenoVue live-cell stains y PhenoVue Fluor 647 live-cell actin stain y PhenoVue Fluor 555 live-cell tubulin stain y PhenoVue Fluor 647 live-cell tubulin stain PhenoVue Fluor 555 live-cell tubulin stain Time-lapse imaging of untreated U20S cells. Image acquisition every 2h for 24h on the Opera Phenix Plus system. 17 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Revvity solutions for live-cell imaging PhenoVue cellular imaging reagents Revvity’s PhenoVue portfolio includes organelle and cell compartment stains, and cell function reagents many of which are compatible with live-cell imaging. Benefits of PhenoVue cellular imaging reagents include: straightforward protocols for ease-of-use, performance demonstrated in biological and pharmacological models, extensive testing to provide optimal formulations and long-term stability and stringent QC for batch-to-batch reproducibility. Cell function reagents and kits y PhenoVue ROS total oxidative stress indicators: ROS-490, ROS-570, and ROS-670 y PhenoVue fluorescent calcium indicators: Cal-520 AM, Cal-590 AM, Cal-520 AM Bright, Cal-590 AM Bright, and Fluo-4 AM y PhenoVue 505 live cell caspase-3/7 activity stain: a no-wash and cellpermeable fluorogenic dye for visualizing apoptotic cells. y PhenoVue live/dead cell viability assay kit: a two-color green/red fluorescence method for the simultaneous determination of live and dead cells. y PhenoVue HypoxiTRAK™: a cell-permeable hypoxia indicator, which exhibits far-red fluorescence. PhenoVue organelle and cell compartment stains y PhenoVue Fluor-Concanavalin A conjugates y PhenoVue Fluor-WGA conjugates y PhenoVue Fluor Lysosomal stains y PhenoVue Fluor Mitochondrial stains y PhenoVue Fluor 493 lipid stain y PhenoVue Nile Red lipid stain y PhenoVue Fluor 512 nucleic acid stain y PhenoVue Hoechst 33342 nuclear stain y PhenoVue DRAQ5™ Time-lapse imaging of U2OS cells stained with PhenoVue 505 live cell caspase-3/7 activity stain in the presence of staurosporine. Images acquired on the Opera Phenix Plus high-content screening system every 5 min for 24 hours. HeLa cells stained with PhenoVue Hoechst 33342 and PhenoVue 503 Lysosomal stain. Imaged on the Operetta CLS high-content analysis system. 18 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Revvity solutions for live-cell imaging From basic research to assay development and screening, Revvity’s high-content imaging systems produce amazing images, so you can take your research further – and make discoveries faster. Both Operetta CLS and Opera Phenix Plus are confocal spinning disk systems, providing gentle imaging with efficient background rejection. y Reduce exposure times with water immersion lenses that improve sensitivity and resolution in XY and Z dimensions, while confocal image acquisition removes much of the light from outside the focal plane. y Maintain optimal cell health with environmental control options for assays running from hours to days. y Analyze live cells gently using label-free digital-phase contrast imaging mode or brightfield imaging. y Perform fast response live-cell assays with dispensing options and fast frame-rate imaging on the Opera Phenix Plus system. Opera Phenix Plus and Operetta CLS high-content imaging systems Opera Phenix Plus high-content screening system Operetta CLS high-content analysis system 19 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Revvity solutions for live-cell imaging The Phenologic.AI module in Harmony and Signals Image Artist™ software harnesses the power of pretrained deep neural networks (DNNs) to provide an efficient and reliable method for identifying cells and cellular nuclei within fluorescent and brightfield images. y Turnkey AI image analysis: Utilize pre-trained AI for easy and efficient analysis of both fluorescent and brightfield images, without re-training by the scientist y Label-free detection: Simplifies detection of cellular nuclei in brightfield images without nuclear staining, saving sample preparation time y Robust identification: Trained on diverse cell lines to reliably identify cellular structures and provides phase contrast imaging for cytoplasm segmentation y Increase multiplexing capabilities: Enhance your experimental flexibility and use more fluorescent dyes with additional markers y Improved viability: Reduce phototoxicity for live-cell experiments by avoiding stress from fluorescent dyes, improving cell viability Phenologic.AI for cellular image analysis Phenologic.AI Time-lapse imaging of MCF7 cells. Brightfield image acquisition every 4 min for 24h on the Opera Phenix Plus system (20x air objective). Phenologic.AI: Nuclei segmentation on brightfield images using a pre-trained Artificial Intelligence (AI) model • Trained on diverse cell line images with varied objectives • Robust segmentation of cells on brightfield images • Pre-trained AI model for multiple magnifications and cell types • Digital phase reconstruction for cytoplasm segmentation 20 | • Introduction • Contact us • Benefits of live-cell imaging • Overcoming the challenges • Revvity solutions for live-cell imaging • Applications of live-cell imaging For research use only. Not for use in diagnostic procedures. Revvity solutions for live-cell imaging The Celigo image cytometer performs live-cell kinetic analysis and cell sample characterization, prior to downstream processing. y Detect, image, and count individual cells in each well y Analyze at the cell level for applications such as: apoptosis, cell cycle, fluorescent reporters, and cytotoxicity y Label-free proliferation y Rapid whole well imaging to help maintain cell health outside the incubator y Automated microplate handling option for kinetic end-point analysis or time-point analysis Assays and applications: y Cell survival y Cytoxicity y Proliferation Celigo image cytometer Celigo image cytometer y Growth curves y Dose response y Cell line expansion y 2D/3D growth inhibition y Tumor spheroid formation Brightfield image of counted cell colonies across a well in a 96-well plate. Brightfield image of the edge of a well in a 96-well plate showing enhanced image quality and contrast. Brightfield image of counted adherent cells in one well of a 96-well plate. www.revvity.com Copyright ©2025, Revvity, Inc. All rights reserved. Revvity is a trademark of Revvity, Inc. All other trademarks are the property of their respective owners. 2060350