Spark^® Cyto

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

LEARN – SELECT – MEASURE

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.

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.

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.