Spark^® Cyto

The Spark Cyto is a multimode reader platform equipped with a highly sophisticated fluorescence imaging module for real-time cytometry.

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Predictive Oncology is a science-driven company offering solutions that introduce human diversity at an earlier stage of the drug discovery process. The company has developed a 3D culture model, r-Breast, to replicate the conditions in the mammary tissue and recreate the tumor microenvironment, including the epithelial and stromal niches. This provides a physiologically-relevant approach to evaluating new drugs or drug combinations, while also accounting for species-specific differences in the extracellular matrix composition. This application note describes a study of two different co-culture systems using breast fibroblasts and T-cells. First, fibroblasts were cocultured with breast cancer cells to assess the impact on drug sensitivity. Second, the effect of programmed cell death protein 1 (PD-1) drug treatment on the viability of breast cancer cells in the presence of T-cells was investigated. Cell viability was assessed using the r-Breast model with propidium iodide-based assays and CellTiter-Glo® luminescence analysis (Promega), efficiently evaluating the results using the Spark Cyto multimode reader.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a global concern, due to its rapid spread. The study, published by the University of Frankfurt, describes the use of the Spark Cyto for the development of a cellular infection model that enables high throughput SARS-CoV-2 experiments and live cell imaging. The automated, non-invasive optical readout included assessment of confluency, roughness factor and fluorescence measurement. The data further highlights the use of the cell line for screening for antiviral compounds as well as for investigating the efficacy of neutralizing antibodies against different SARS-CoV-2 variants.

This application note systematically defines experimental parameters to enable live cell nuclear staining with minimal cytotoxicity during repeated exposure and continuous fluorescence imaging in the Spark Cyto, as well as robust automated cell segmentation with the SparkControl™ and Image Analyzer™ software.

The optimized procedure was used in a multiplexed, three-color assay to detect and distinguish early and late stages of apoptotic cell death, opening the door for dynamic long-term phenotype tracking.

Alzheimer’s disease (AD) and vascular dementia (VaD) are the two most common types of dementia, and their incidence is increasing year by year. They affect the health of the elderly, and cause a huge burden on society, with no effective treatments currently available.

In vitro research is the first step in drug evaluation, and cellular immunofluorescence can be used to more clearly define the role of drugs, provide more comprehensive and accurate information for downstream drug development.

This study uses the Spark Cyto – an innovative multimode reader with cell imaging capabilities – to evaluate the neuroprotective effects of miR-23b-3p (miRNA) and tilianin (compound) on stable transfected cell lines and human primary neurons. The results in this application note are based on a peer-reviewed article, published in the journal Oxidative Medicine and Cellular Longevity in August,2021, with the title: Tilianin Ameliorates Cognitive Dysfunction and Neuronal Damage in Rats with Vascular Dementia via p-CaMKII/ERK/CREB and ox-CaMKII-Dependent MAPK/NF-κB Pathways.

Scientific innovation is dependent on the power of observations, and the strength of the conclusions drawn from those observations. In in vitro biology, a majority of physiologically relevant outcomes adhere to dose- and exposure-dependent factors. Unfortunately, data collection for traditional cell-based experiments often occurs at arbitrary but convenient endpoints, and using inadequate or poorly informative tools. The resulting data typically lack the appropriate kinetic resolution to fully answer details of the cause-and-effect relationship. Because biological pathways and processes are already inherently complex, a more efficient screening paradigm is required, allowing scientists to examine the important parameters of ‘when’ and ‘how’ to better characterize a given response. This application note seeks to provide a practical demonstration of how real-time assays and a plate reader with bright field and fluorescence imaging functionality can work in unison to reveal cell- and compound-specific features of an apoptotic response.

This application note describes the outcome of an experiment comparing the Opera Phenix with the Spark Cyto for the detection of apoptotic cells. For this study, Hoechst 33342 was used as a ‘blue’ nucleic counter stain, with TMRM and YO-PRO-1 as the two secondary mask signals. Please note that findings presented here do not necessarily reflect the general performance difference between the two systems.

Workflow automation with the Spark Cyto multimode reader.

Automated monitoring of the reporter activity of hfob.1.19 cells following induction of osteogenic differentiation by temperature shift

While 3D cultures are superior to the 2D culture approaches in terms of physiological tissue organization and providing robust prediction of clinical outcomes, these techniques are often more expensive and time consuming to perform compared to the standard 2D protocols. Performing multiplexed assays with various readouts is therefore beneficial in order to gain the most information from each experiment. Here, we describe a multiplexed approach combining sequential imaging based LIVE/DEAD cell viability assays (Thermo Fisher Scientific) and CellTiter Glo luminescence analysis (Promega) using the Tecan Spark Cyto with r-Breast and r-mBreast models.

The genomic integrity of mammalian cells is constantly challenged by DNA damage arising from both endogenous and exogenous sources. Complex DNA repair pathways have evolved to deal with specific types of DNA lesions. An efficient DNA damage response (DDR) requires immediate detection and repair of the damage.

In addition, cell cycle checkpoints need to be activated to allow enough time for repair, prevent further damage through collision of the replication and transcription machinery with unrepaired lesions, and ensure that lesions are not passed on to daughter cells.

While impaired DDR can lead to serious diseases like cancer, it also presents an opportunity for therapeutic interventions exploiting these repair defects. 1 DNA repair is a highly dynamic process involving distinct and well-coordinated steps, and should therefore ideally be studied in living cells.2 However, a lot of post-translational modifications essential for the DDR can only be analyzed in fixed cells.

Highly aggressive cutaneous squamous cell carcinomas (SCCs) occur particularly frequently and early in patients suffering from the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB), causing a high mortality rate...

Predictive Oncology’s r-Tube 3D model mimics the niches in which endothelial cells reside within tissues, providing a more physiologically accurate platform. This allows the effectiveness of novel drugs or drug combinations to be evaluated in a setting that captures the species-specific differences in ECM composition. In this manner, the r-Tube 3D system offers a valuable tool for studying cancer and contributing to the development of improved therapeutic strategies, by incorporating the microenvironment into the culture model. The Spark Cyto has been widely used for bright field and fluorescence imaging in 3D models and has proven suitable for imaging thick samples with cells embedded in a 3D matrix. A multiplex approach using the r-Tube 3D model to investigate eribulin and ramucirumab treatment was therefore performed using the Spark® Cyto with sequential bright field imaging and CellTiter- Glo® luminescence analysis (Promega).

The essence of 3D liver tissue reconstruction lies in creating functional liver tissue outside of the body, mimicking the native ECM and microenvironment. This approach offers several advantages. First, it enables the study of liver pathophysiology and disease mechanisms, leading to better understanding of novel therapeutic targets. From a regenerative medicine standpoint, 3D liver tissue reconstruction offers patient-specific solutions; patient-derived tissues can be created using primary liver cells, minimizing the risk of immune rejection and eliminating the reliance on donor organs. These engineered liver tissues hold great promise for transplantation and tailored treatment plans. Furthermore, 3D liver tissue models provide an ethical and clinically relevant platform for drug testing, reducing the need for animal testing. This application note describes the combination of Tecan’s Spark Cyto reader with Predictive Oncology’s r-liver-tox set-up to perform multiplexing investigations, including live/dead cell analysis, detection of intracellular production of reactive oxygen species (ROS) and the immunostaining of primary human hepatocytes.

Predictive Oncology has developed a three-dimensional (3D) model called the Reconstructed Pancreas (r-Pancreas), which attempts to replicate the pancreatic tumor microenvironment. It successfully recreates the conditions found in pancreatic tissue – including both the epithelial and stromal niches – offering a more physiologically relevant approach to assess new drugs, or combinations of drugs. As described in this Application note, this research model was used in combination with the Spark Cyto multimode reader to investigate the efficacy of gemcitabine, a therapeutic agent against pancreatic tumor cells. To enhance the physiological relevance of the r-Pancreas model, a collagen-rich capsule was introduced, as human pancreatic tumors are known to possess an outer layer of stiff ECM, acting as a physical barrier that hinders drug penetration. Moreover, by incorporating a co-culture of primary activated pancreatic stromal cells and pancreatic tumor cell lines, it was possible to create a more comprehensive 3D model that allows testing of potential therapeutic agents within the pancreatic tumor microenvironment. In this model, pancreatic tumor cells were co-cultured with human pancreatic fibroblasts.

Streamlining 3D cell culture-based drug response profiling with the Fluent® Automation Workstation, D300 Digital Dispenser, and Spark® Cyto multimode reader.

The development of treatments for snake bite induced vascular toxicity relies on robust, quantitative, and relevant in vitro models of blood vessels. Organ-on-a-chip is an emerging technology that uses miniaturized components to create a three-dimensional culture of human cells grown in a microfluidic system. In contrast to cells grown in 2D, blood vessels grown in microfluidic channels enable the inclusion of several important physiological parameters during cell culture, such as 3D tubular morphology, fluid perfusion, inclusion of extracellular matrix (ECM) and exposure to biochemical gradients. Real-time assays combining organ-on-a-chip platforms with multimode readers would enable direct monitoring of vascular barriers, viability and morphology with a sufficient temporal resolution to capture the fast effects of potent snake venom toxins. This application note describes the use of the Spark Cyto to multiplex readouts and comprehensively assess the potential of different snake venoms to cause vascular toxicity of a human blood vessel model grown in an organ-on-a-chip platform.

Patient-derived organoids (PDOs) are in vitro models originating from normal or cancerous stem cells that preserve the original cellular complexity, tissue morphology and pathophysiology. Currently, working with PDOs can be challenging due to the lack of automated equipment suitable for performing high throughput, imaging-based drug screening in organoid analysis pipelines. Tecan is addressing this need with a deep learning-based algorithm for the Spark® Cyto multimode plate reader, allowing automatic identification and segmentation of objects with complex 3D structures, such as organoids or spheroids. This application note evaluates the Spark Cyto for automated PDO imaging and analysis, using the new 3Dai deep learning-based algorithm available in the SparkControl™ software, as well as optimization of settings and re-analysis of the images in the Image Analyzer™ software.

Analyzing the dose-response relationship using cell-based assays is essential to understanding a drug’s efficacy. However, fixing the appropriate dose range and time point is often challenging. The D300e Digital Dispenser enables accurate delivery of compounds at picoliter levels, spanning a wide dose range – for example, from 0.2 nM to 1 μM – in just a few minutes. This can be combined with the live-cell imaging and full cell incubation capabilities of the Spark Cyto multimode reader to monitor cell proliferation under standard culture conditions (37 ºC, 5 % CO2). This technical note uses the D300e and Spark Cyto to comprehensively capture an anti-cancer drug’s efficacy profile, tracking cell viability with nine doses at 25 time points using luminescence-based and bright field imaging technologies simultaneously.

This Technical Note briefly describes how to use Image Analyzer for the optimization of confluence assessments in bright field images, as well as for object segmentation and counting in fluorescence images.

This application note describes a semi-high throughput approach for the analysis of intracellular Ca2+ and cellular Ca2+ uptake using the Spark Cyto’s multicolor fluorescence imaging and the Image Analyzer™ software’s integrated Voronoi analysis function. Treatment with the calcium ionophore ionomycin was used to increase intracellular Ca2+ concentrations, and Fluo-4 was used in combination with the nuclear counterstain Hoechst 33342 for measurement quantification.

This technical note describes the capabilities of the Spark Cyto to normalize a cellular signal to the cell number, or cell mass, per well. This dramatically improves the overall data quality of the respective cell-based assay and, finally, leads to an improved reproducibility of cell-based experiments and more efficiency in the lab.

Fluorescence imaging using the spark cyto – acquisition in a snap using sparkcontrol™

Accurate and automated assessment of cell density using bright field and fluorescence imaging…

Automating visualization and quantification of protein expression in microplates…

Discriminating viable, apoptotic and necrotic cells.

Quantification of life: Dead less ratio with the Spark Cyto imaging cytometer.

SparkControl™ offers a wide selection of predefined plate definition files (.pdfx), and it is crucial to use the correct plate definition for imaging applications to avoid autofocus errors. Alternatively, the Plate Geometry Editor can be used to modify existing files to suit unlisted plate formats.

This Technical Note describes the use of the Plate Geometry Editor in SparkControl™ and points out the most important aspects for the successful creation of pdfx files.

A non-invasive way to accurately count cells in brightfield using a deep learning algorithm.

Cell line development plays a crucial role in modern biological research and drug development. Clonal lines derived from a single cell can provide a reproducible and stable platform for drug discovery, studying biological processes and producing recombinant proteins. Genetic engineering and genome editing techniques have enabled the creation of cell lines with specific mutations or gene knockouts, allowing researchers to investigate the function of individual genes and pathways. Additionally, cell lines are used in the production of biologics, such as monoclonal antibodies and recombinant proteins, making the development of new and improved cell lines a critical area of research in both academia and industry. This technical note describes a method for using the Spark Cyto multimode reader to perform whole-well imaging of single mammalian cell clones in 96-well plates, in order to identify optimal clones. Both the Spark and Spark Cyto are capable of verifying monoclonality, however the Spark Cyto offers a faster readout and improved image quality compared to the Spark’s basic imaging functionality, providing higher throughput clone identification.

This technical note describes the use of the Uno Single Cell Dispenser to isolate single cells. This instrument harnesses microfluidic digital dispensing technology to gently isolate viable cells for various downstream applications, including cell line development.

The viability of the single cell post dispensing was assessed by addition of a fluorescent dye, that bound to the DNA of cells with impaired membrane integrity, using whole-well fluorescence imaging functions of the Spark Cyto.

Cell outgrowth rate was determined 10 days after single cell isolation with the Uno, using whole-well brightfield imaging. Using Uno and Spark Cyto together, enables simple single cell isolation and determination of monoclonal cell outgrowth for variety of downstream application.

Recent advancements in cell-based research strategies have revolutionized drug discovery, disease modeling, tissue engineering, and personalized medicine. The shift from traditional 2D monolayer culture to three-dimensional (3D) cell culture systems has opened new avenues for more realistic representation of complex tissues within the human body. Particularly the use of multicellular 3D spheroids is showcased here. These spheroids represent a significant advancement in cell-based assays. In this note, the generation of spheroids in Corning 96-well, round bottom ULA microplates and subsequent monitoring and analysis of spheroid growth using the Spark Cyto multimode reader is presented. The integration of a plate washer (HydroSpeed™) further optimized the workflow, enhancing automation, standardization, and reproducibility, while reducing human errors. These advancements represent a substantial step forward in enhancing our understanding of cellular behaviors and developing more realistic in vitro models for various applications.

Traditional two-dimensional (2D) cell culture models, wherein cells are cultivated on planar and rigid substrates, exhibit limitations in predicting clinical trial outcomes due to the disparity in cellular behavior compared to in vivo conditions. In contrast, three-dimensional (3D) cell culture models, such as tumor spheroids and organoids, have gained prominence as they more accurately mimic the tumor microenvironment, thereby providing enhanced predictive validity for drug response and activity. However, the increased physiological relevance of these 3D models introduces challenges related to experimental and technical complexity. This technical note delineates the workflow for generating multiple spheroids embedded within a Matrigel matrix and discusses the application of z-stack imaging coupled with artificial intelligence (AI)-based automated analysis for the segmentation and evaluation of spheroids in the brightfield channel.

Staining and fixing cells or cellular aggregates for endpoint analysis is a labor-intensive process in cell biology, traditionally performed using manual protocols that involve multiple incubation and washing steps, aiming to enhance the microscopic visibility of specific cellular compartments. In 3D cellular aggregates, optimizing staining and washing protocols is essential to ensure even reagent distribution and minimize variability. However, manual handling often introduces procedural errors, compromising the quality and reliability of experimental results. This technical note highlights the integration of Tecan's HydroSpeed™ plate washer into the staining and fixation workflow for 3D multicellular spheroids. The automated system improves throughput, standardizes washing procedures, and reduces well-to-well variability, while minimizing cell loss, making it highly suitable for high-throughput screening and drug discovery applications.

In this Technical Note, we describe how to use brightfield imaging and label-free counting analysis with the Spark Cyto to analyze the solubility of drugs in 96- and 384-well plates. Results are compared to those generated with the BMG Labtech NEPHELOstar Plus, a microplate reader offering nephelometric readout capabilities. Data presented in this study show that the results acquired with the imaging-based approach closely match those of the standardized nephelometric approach, differing by a maximum of 10%. The images generated by the Spark Cyto clearly add another layer of confidence to solubility assessments. The main benefit is that suspect wells can be easily investigated through visual inspection of the images by the user, allowing for easier identification of possible false positive or negative results in combination with the nephelometric readout.

Bringing time and cost advantages to labs…

Cell-based assays have a variety of practical uses within scientific research and are an integral component of many scientists’ workflows from testing tumor resistance for cancer research to helping identify novel therapeutic compounds in drug discovery.