Experimental assays

We specialize in cell-based assays that utilize a variety of experimental models, cell culture conditions, detection methods, and readout platforms. This allows us to measure important cellular parameters under a wide range of experimental conditions. Our assay results can be analyzed using various statistical and analytical tools to provide meaningful insights and make informed predictions. Our team of experts is dedicated to providing high-quality and reliable services to meet your research needs.

Selected preconfigured assays are listed and described below.

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Cell health assays

Cell health assays measure various cellular processes to provide insight into how and why cells proliferate or die. These are primary tools to assess the cytotoxicity of drug candidates or other compounds, analyze cellular parameters in various diseases including cancer, and study basic cellular processes.

The selection of a specific cell health assay depends on the parameters that need to be measured, the required sensitivity, the throughput, and the model system. Three main classes of cell health assays can be distinguished:

• viability assays that detect the number of living cells
• cytotoxicity assays that detect the number of dead cells
• apoptosis assays that evaluate the mechanism of cell death

Depending on the required sensitivity, the throughput, and the model system, various assay formats, signal detection techniques, and devices can be used, including:

• Fluorescence, luminescence, or colorimetric assays compatible with multi-mode microplate readers
• Stain-free or fluorescence intensity microscopy imaging for cell counting
• Cell labeling with specific live/dead cell dyes and apoptotic markers for flow cytometry

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Cell viability

Cell viability assays measure the relative number of live cells in cell populations, in response to a drug or other perturbation, by involving different experimental techniques depending on the required sensitivity, the throughput, and the model system. Viability assays can be multiplexed with toxicity and cell death assays to provide additional information regarding the relative number of dead cells and type of cell death. We offer assays suited for a broad range of applications that are based on bioluminescent, fluorometric, or colorimetric readouts. Endpoint assays provide sensitive, high-throughput formats, whereas real-time, live-cell assays repeatedly monitor over time and generate multiple data points from a single assay.

The essential cell viability assays are summarised in the table below: 

In addition to the above most commonly used assays we also offer alternative approaches to suit specific experimental needs, contact us for more info.

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Cell toxicity

One of the main events that occur after cell death is the loss of membrane integrity, which allows chemicals or proteins to freely enter or exit the cell. Taking advantage of this phenomenon, we are able to evaluate the cytotoxicity of a given compound either by determining the exact number of dead cells stained with cell-impermeant fluorescent dyes or indirectly, by measuring the activity of enzymes that leak into the extracellular medium after cell membrane damage.

Membrane integrity dyes are cell-impermeant and only able to enter cells with a compromised plasma membrane. We use the nucleic acid-binding dyes (eg. DRAQ7, PI, 7-AAD, EthD-1, SYTOX) which are nonfluorescent in aqueous media but exhibit increased fluorescence upon binding to double-stranded DNA (dsDNA) or RNA. They allow for the fast and accurate evaluation of cytotoxicity and for multiplex experiments with other fluorescent dyes.

Enzyme leakage-based cytotoxicity assays detect extracellular glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH) or adenylate kinase (AK) released from damaged cells. Released enzymes activity can be quantified by a coupled enzymatic reaction in which provided substrates (lactate and NAD+, NADP+ or ADP) are converted to NADH, NADPH or ATP which further can be measured using different assay chemistries. The generated color, fluorescent or luminescent signal is proportional to the amount of ezyme present in analysed sample. There is no need for cell lysis so repeated samples of supernatant can be taken from wells over time without disrupting the cells themselves. This allows for kinetic analysis of cell death and multiplexing with other tests.

The essential cell toxicity assays are summarised in the table below:

In addition to the above most commonly used assays we also offer alternative approaches to suit specific experimental needs, see more info.

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Cell death

Cell-death assays are crucial tools for drug discovery and development, providing insights into the mechanisms of programmed cell death and drug-induced cell death. By using these assays, researchers can assess the efficacy and safety of potential drug candidates, optimize drug dosages, and identify potential adverse effects.

Understanding the type of cell death induced by a drug candidate is important for drug candidate profiling because different types of cell death have different mechanisms and implications for drug efficacy and safety.

The commonly used cell death assays are summarised in the table below:

In addition to the above assays we also offer alternative approaches to suit specific experimental needs, see more info.

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Cell functional assays

Functional assays are used to monitor and evaluate key cellular processes, like metabolism, cell-cycle progression, cellular export/import, motility, and others. The diversity of these assays reflects the diversity of cellular processes. Functional assays are used for many different applications including the impact of drug candidates or other compounds on different cellular functions, and analysis of cellular changes in cancer or other diseases. Our portfolio of functional assays offers a range of solutions suited to measure different cellular processes, in different model systems with the required sensitivity and throughput.

Mitochondria function assays

In spite of the critical roles that mitochondria play in all cells and the many ways they can be adversely affected by chemical compounds, mitochondrial toxicity might be crucial to identify during drug testing. Mitochondria can be affected by drug treatment, resulting in cardio- and hepatotoxic side effects that can lead to drug withdrawal from the market. In fact, the FDA suggests that, for example, all antiviral drugs should be tested for impact on mitochondrial function. A number of different methods can be used to monitor and evaluate changes in the morphology and function of the mitochondria.

ATP depletion assay

Mitochondria produce most of the cellular energy in the form of adenosine triphosphate (ATP) via oxidative phosphorylation. Many cell lines are metabolically adapted to growth in high glucose media and derive most of their energy from glycolysis rather than mitochondrial oxidative phosphorylation which reduces the susceptibility of the cells to mitochondrial toxicants. Using galactose as an energy source cells are forced to rely on mitochondrial oxidative phosphorylation for ATP production. To detect mitochondrial impairment we compare the toxic effects of different drugs on mitochondrial ATP production capability in the glucose and galactose media. For the measurement of ATP level we use assays based on luciferase activity and luminescence readout. In addition to the standard endpoint measurement of ATP in lysed cells, we are able to perform kinetic measurements in living cells.

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Measurement of mitochondrial membrane potential 

The electrochemical gradient across the inner mitochondrial membrane (mitochondrial membrane potential) is the driving force behind oxidative phosphorylation and its maintenance is critical for normal cellular function. Mitochondrial membrane potential as an integral component of cellular energy homeostasis is therefore a reasonable in vitro indicator of chemically induced cytotoxicity. We use a number of fluorescent dyes to accurately measure mitochondrial membrane potential. Tetramethylrhodamine methyl ester (TMRM), rhodamine123 or MitoTrackers passively diffuse across the plasma membrane and accumulate in active mitochondria generating single-component fluorescence signals. In contrast to these monochromatic probes, the ratiometric dyes like JC-1 and its derivatives (eg. Mito-ID) exhibit potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm) with increasing concentration (i.e., aggregation). The ratio of green to red fluorescence depends only on the mitochondrial membrane potential and not on other factors such as mitochondrial size, shape, and density.

Analysis of mitochondrial mass and morphology

Mitochondrial mass (amount of mitochondria per cell) can be the measure of mitochondrial toxicity. An increase in mitochondrial mass is thought to occur as a consequence of an adaptive response by the cell to increase energy production on the other hand mitochondrial damage can also manifest itself as a decrease in mitochondrial mass in the cell. Changes in the mitochondrial mass are often preceded by mitochondrial morphology transitions regulated by dynamic membrane fusion and fission processes. Both mitochondrial mass and morphology changes can be monitored in single cells with the use of fluorescent probes targeting mitochondria regardless of mitochondrial membrane potential.

Oxidative stress

Mitochondria are considered a primary intracellular site of reactive oxygen species (ROS) generation. ROS are intimately involved in redox signaling and have a role in a number of cellular processes, but in some situations can also lead to oxidative damage. Oxidative stress occurs when there is an increased production of reactive oxygen species (ROS) or a decrease in the effectiveness of cellular antioxidant defenses. Assays to determine oxidative stress may measure levels of toxic reactive oxygen species or levels of cellular antioxidants (e.g. glutathione). We analyze the oxidative stress by measuring the generation of ROS in mitochondria as well as in the whole cell with the use of different fluorescent or luminescent probes. The level of oxidised form of glutathione (GSSG) or the ratio of reduced and oxidized forms (GSH/GSSG) is also an excellent indicator of oxidative stress in cells.

Activity of OXPHOS complexes

The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I–V proteins in the ETC. The respiratory chain complexes are important for drug discovery in that they can be involved in toxic effects or might be of interest as drug targets themselves. We measure the inhibition of each complex activity in the presence of test compounds using MitoTox colorimetric activity assays.

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Absorption and permeability

The permeability of compounds across cell membranes (e.g., intestinal epithelium) is a critical characteristic that determines the rate and extent of absorption and ultimately affects the bioavailability of a drug candidate. We provide bidirectional permeability assays in human Caco-2 and MDCK cells.

Parallel Artificial Membrane Permeability Assay (PAMPA)

Drugs often need to cross cell membranes in order to reach their target of action and this makes a compound’s ability to passively cross these membranes an important characteristic to evaluate. The Parallel Artificial Membrane Permeability Assay (PAMPA) is used as an in vitro model of passive, transcellular permeation. The assay allows for measuring the gastrointestinal permeability of oral therapies, blood-brain barrier permeability, and dermal/transdermal penetration potential.

Caco-2/MDCK permeability assay

The Caco-2 permeability assay utilizes Caco-2 cell line derived from human colon carcinoma, which has many characteristics that resemble intestinal epithelial cells. This assay offers a measure of the permeability across the intestinal barrier in both directions: the compound is added to the apical or basolateral compartment and efflux across the monolayer of cells is monitored. The amount of compound that has permeated across the cells is measured by LC-MS/MS.

The Madin-Darby Canine Kidney (MDCK) cell is an epithelial cell line derived from the canine kidney. The expression of transporter proteins and metabolic activity are low for MDCK cells but compared to Caco-2 cells, they proliferate and differentiate more quickly. Hence, it becomes an attractive alternative assay compared to Caco-2 permeability assay to assess the human intestine barrier.

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Cell migration and invasion assays

Metastasis is the cumulative result of multiple changes in tumor cells and their microenvironment that enables cellular migration and invasion into healthy host tissue. Cell migration assays enable quantitative characterisation of cells movements, adhesion, and invasion and how these are influenced by pharmacological agents. We use different techniques to study these phenomena, from endpoint assays to time-lapse microscopy approaches and complex analysis for the downstream interpretation of the cell migratory behavior.

Wound healing assay

The wound-healing assay is a simple and inexpensive method to study directional cell migration in vitro. The basic step involves creating a “wound” in a cell monolayer manually or utilizing special microplates that provide a uniform and reproducible cell-free zone (i.e. Oris^™ Pro). Cells are then imaged at the beginning and at regular intervals during cell migration. It is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration. This assay is convenient and versatile and can be applied for a high throughput screen platform.

Transwell cell migration/invasion assay

The most widely accepted cell migration technique is the Boyden Chamber assay. The classic transwell migration assay system uses a hollow plastic chamber, sealed at one end with a porous membrane. This chamber is suspended over a larger well that contains medium and/or chemoattractants. Cells are placed inside the chamber and allowed to migrate through the pores, to the other side of the membrane. To analyze cell invasion, the transwell insert membrane is coated with basement membrane ECM protein or a layer of cells such as endothelial cells. Migratory cells are then stained and directly counted in the microscope or some other indirect readout methods are used (e.g. metabolic activity assay).

Microfluidic migration device chemotaxis assay

The alternative for the transwell system are microfluidic migration devices which promote a stable diffusion-generated concentration gradient. Slides are made from plastic with high optical qualities similar to those of glass allowing for life-microscopy assays. At specific time intervals, images of the observation area can be acquired, allowing real-time monitoring and quantitative measurements of cell migration.

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HCS/HTS screening

High-content screening (HCS) is a powerful tool that uses microscopic imaging to assess multiple cellular parameters within a single bioassay. This approach is highly efficient and can be seamlessly integrated into high-throughput screening (HTS) by utilizing various multiwell plate formats, including 96, 384, or 1536 wells. By employing fluorescent dyes, fluorescently labeled proteins, and antibodies, we can accurately detect and measure cellular changes under diverse conditions, such as exposure to different compounds or varying doses.

Partner with us to unlock the full potential of HCS in your research and gain fresh insights into cellular processes.

After capturing images, our advanced image processing tools extract specific features, providing valuable quantitative data. The number of features we can quantify is extensive, allowing us to reveal multiple layers of information tailored to your research objectives.

For example, we can use a DNA-binding dye to assess nuclear size, shape, intensity, and texture. We can also track changes within a cell by monitoring a DNA-binding dye to identify the nucleus and then measuring nuclear translocation of fluorescently labeled protein in response to a stimulant. Our fluorescence-based HCS assays often incorporate reporter cell lines, where fluorescently tagged proteins help quantify protein localization, translocation, levels, and colocalization.

Alternatively, after fixing the cells, we can perform immunofluorescence for specific proteins of interest. Additionally, we utilize function- or organelle-specific dyes to quantify features such as oxidative stress, glutathione levels, mitochondrial function, nuclear shape, chromatine condensation, and cell cycle dynamics. Data can be reported either for individual cells or as well-averaged results.

HCS offers a wide array of applications, making it an invaluable tool for various research studies:

• Protein Dynamics: Explore changes in protein levels and localization within individual cells, such as transcription factors or signaling proteins in the nucleus, cytoplasm, or other cellular subcompartments.
• Transcriptional Insights: Monitor transcriptional responses in individual cells using innovative techniques like fluorescent-reporter cell lines or single-molecule RNA-FISH.
• Proliferation and Cell Cycle Studies: Accurately quantify cell proliferation within co-culture settings, allowing for in-depth studies such as targeted elimination of cancer cells and the assessment of the cytostatic effects of various drugs.
• Morphological Analysis: Perform detailed quantification of cell morphology parameters, gaining valuable insights into cellular shapes and interactions.
• Subcellular Profiling: Profile subcellular compartments with precision, examining aspects such as protein and mRNA localization as well as organelle morphology.
• Real-Time Imaging: Capture dynamic cellular events in real-time, including phenomena like Ca2+ fluxes (using indicators like Fluo 4) and apoptosis (detected through methods like pSIVA)

Our HCS/HTS microscopy services include the following assays.

Cell counting

Cell counting assays are commonly used in high-throughput microscopy screening enabling the precise determination of cell numbers within a sample. This method involves the use of fluorescent dyes that selectively label the cell nuclei or other cellular components, allowing them to be easily visualized and counted under a microscope.

The basic steps of a typical cell counting assay include:

• Sample Preparation: Cells are cultivated in suitable media and subjected to specific compounds or conditions, tailored to the experiment’s requirements.
• Cell Labeling: The introduction of fluorescent dyes or stains that target cellular components like DNA, RNA, or proteins is a critical step. Common choices include DNA intercalating dyes such as Hoechst, DAPI, or SYTOX, which stain cell nuclei.
• Imaging and Analysis: High-throughput microscopy captures images of the labeled cells. Specialized software then automatically segments and counts the cells within each sample.

Cell counting assays find applications across various domains, including drug discovery and cytotoxicity testing. They are particularly useful in high-throughput screening, where large numbers of samples need to be analyzed quickly and efficiently. In addition to conventional cell culture experiments, we offer co-culture assays. These assays recreate a more physiologically relevant microenvironment for drug testing. Notable examples include assessing the impact of immune cells on cancer cell growth and exploring the influence of microenvironments generated by specific cell types (e.g., fibroblasts on cancer cells).

Assay summary

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Cell cycle analysis

The Cell Cycle Analysis Assay is a vital tool for assessing the effects of various compounds and treatments on the cell cycle. This assay provides valuable insights by measuring changes in DNA content, synthesis, and the number of actively dividing cells. It plays a crucial role in understanding how different agents influence the intricate dynamics of the cell cycle, making it an essential component of our research services.

This comprehensive assay involves the following key steps:

• Incorporation of EdU: To detect newly synthesized DNA, we employ a fluorescently labeled nucleoside analog, 5-ethynyl-2′-deoxyuridine (EdU), providing real-time insights into DNA replication.
• Fluorescent Labeling: Cells are carefully labeled with DNA-binding fluorescent dyes, including widely used options like propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), or Hoechst.
• Identification of Actively Dividing Cells: Immunostaining targeting phosphohistone H3 further enhances our capabilities. It allows us to identify cells that are actively undergoing division.
• Fluorescence Microscopy: The labeled cells are subjected to fluorescence microscopy, enabling us to precisely analyze their DNA content and synthesize invaluable data about their cell cycle stage.

Cells navigate through distinct phases during the cell cycle, encompassing G1, S, G2, and M phases. Our approach involves measuring the relative distribution of cells across these phases, providing valuable insights into how specific treatments impact cell cycle progression.

For instance, when assessing the effects of a treatment, we examine the proportion of cells in each phase. A compound that inhibits cell proliferation may lead to an increased presence of cells in the G1 phase. In contrast, a compound that stimulates cell growth could result in a higher proportion of cells in the S phase. Our methodology empowers us to precisely evaluate and quantify these changes using high-throughput microscopy to screen large libraries of compounds or drugs, facilitating a deeper understanding of treatment outcomes.

Assay summary

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Protein levels

The Multiplexed Protein Levels assay is a type of immunostaining procedure used in high-throughput microscopy screening to analyze multiple protein levels in a single sample using sequential immunostaining protocols. This assay is also known as Iterative Indirect Immunofluorescence Imaging (4i) or Cyclic Immunofluorescence (CycIF).

The assay involves the following steps:

• Immunostaining: Cells are labeled with specific antibodies, each tagged with a different fluorophore, allowing for precise detection of multiple proteins of interest.
• Fluorescence Microscopy: The sample is imaged using fluorescence microscopy, and the fluorescence intensity emitted by each fluorophore is measured.
• Iterative Analysis: The strength of this assay lies in its iterative nature. After each round of imaging, the previously used antibodies are either removed or antibody-conjugated fluorophores are bleached. New antibodies are then introduced to enable the analysis of additional proteins of interest.

This versatile assay is widely applied in screening large libraries of compounds or drugs to assess their impact on cellular protein levels. It is a valuable tool for identifying potential candidates in cancer therapies and various other applications, offering a comprehensive view of complex cellular signaling pathways and protein interactions.

Assay summary

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Cell morphology

Within our Cell Morphology Analysis, we navigate the complex realm of cell behavior and function, offering a profound exploration into how prospective drug candidates influence the structural and morphological aspects of living cells. This assay involves a systematic approach:

• Compound Treatment: Live cells are subjected to diverse compounds or drugs, each holding the promise of revealing unique insights into cellular responses.
• Cell Staining: To visualize specific cell features, we utilize selective cell staining techniques. Various dyes, molecular probes, or antibodies are employed to highlight specific cellular components, aiding in the comprehensive understanding of cellular changes.
• Automated Microscopy: High-content screening with automated microscopy ensures precise imaging by utilizing appropriate magnification for optimal resolution. This approach allows to capture of the dynamic changes in treated cells, delivering a comprehensive visual dataset of the evolving cellular landscape.
• Image Analysis: Cutting-edge image processing software then steps in to quantify an array of essential morphological features, including:
  
  – Cell size (area, perimeter, diameter)
  – Cell shape (circularity, aspect ratio, form factor)
  – Cell texture (granularity, roughness)
  – Nucleus size and shape (area, circularity, eccentricity)
  – Nuclear-cytoplasmic ratio
  – Cell polarity (polarization index, directional orientation)
  – Filamentous structures (number, length, thickness)
  – Mitochondrial morphology (area of the mitochondrial network, number of branches, shape)
  – Endoplasmic reticulum morphology (number of fragments, length, complexity)
  – Golgi apparatus morphology (number, size, distribution)

The Cell Morphology Assay serves as a valuable window into the effects of drugs, shedding light on cellular signaling pathways, cytoskeletal organization, and cell-cell interactions.

Through high-throughput microscopy, we rapidly screen expansive compound libraries, pinpointing those with the potential to induce the desired changes in cell morphology, a promising indicator of their therapeutic potential.

Assay summary

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Transcript levels

Our state-of-the-art Single-Molecule RNA Fluorescence In Situ Hybridization (smRNA-FISH) service offers an indispensable approach to studying individual gene transcripts within single cells. This sophisticated technique involves the following key steps:

• Cell Cultivation: Cells of interest are cultured within multiwell plates (96 or 384 well) for defined periods, creating an environment conducive to detailed cellular exploration.
• Biochemical Investigations: We afford the flexibility to introduce a spectrum of biochemical interventions. This may involve the application of compounds of interest, thoughtfully aligned with the specific objectives of your research.
• Incubation and Fixation: Following the prescribed incubation period, the fixation process halts cellular processes, ensuring the molecular integrity of the specimens for subsequent analysis.
• Fluorescent DNA Probes: The core of smRNA FISH methodology involves the utilization of custom-designed, fluorescently labeled DNA oligonucleotide probes. The application of multiple probes per mRNA molecule as well as signal amplification methods significantly heightens the sensitivity of the assay, enabling the precise detection of mRNA expression. Our approach accommodates a dynamic range, from individual transcript molecules to several thousand, delivering a comprehensive view of gene expression at the single-cell level.

smRNA-FISH service represents a breakthrough in single-cell gene transcript analysis, shedding light on the intricacies of gene expression dynamics and contributing to a deeper understanding of cellular function and molecular processes. Notably, smRNA-FISH offers distinct advantages compared to traditional RNA-seq and qPCR methods:

• Spatial Mapping: Beyond mere quantification, our technique offers crucial insights into the spatial distribution of transcripts within the cell. This spatial context enriches our comprehension of gene function within distinct cellular locales.
• Multiplexing Capabilities: By fine-tuning the protocol for immunofluorescence staining, we enable the harmonious integration of smRNA-FISH with the concurrent detection of proteins of interest within the same single cell. This multifaceted approach unlocks the ability to scrutinize mRNA and protein expression simultaneously, uncovering intricate correlations between genetic and protein factors.
• Single-Cell Resolution: Unlike bulk RNA-seq techniques, smRNA-FISH provides single-cell resolution, allowing the examination of gene expression at the individual cell level which s crucial for identifying cell-to-cell variations and heterogeneity within a population.
• High Sensitivity: smRNA-FISH is highly sensitive and can detect low-abundance transcripts, making it a valuable tool for studying genes with low expression levels in single cells.
• Complementary Technique: smRNA-FISH can be used in conjunction with single-cell RNA-seq and qPCR to provide comprehensive insights into gene expression at different levels, offering a well-rounded approach to gene expression analysis.

Assay summary

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Live microscopy

Live microscopy is an advanced research tool that allows scientists to study cellular processes in real-time, providing a deeper understanding of dynamic events that might be missed in traditional end-point assays. With the ability to visualize and quantify transient events, as well as fast-kinetic events such as calcium fluxes, and the ability to run long-term time-lapse assays to monitor processes like apoptosis induction, live microscopy offers a wide range of applications for researchers.

Automated live cell imaging systems enhance this technology by enabling continuous monitoring of cells over extended periods of time. This allows researchers to uncover a wealth of cellular dynamics parameters, making live microscopy particularly useful in early-stage drug development. By gaining a deeper understanding of the dynamic nature of cells in-vivo, researchers can identify potential therapeutic targets and develop more effective treatments.

Some specific services that can be provided through live microscopy include:

• Visualization and quantification of transient events, such as dynamic signaling responses
• Study of fast-kinetic events such as calcium fluxes
• Long-term time-lapse assays to monitor processes such as apoptosis induction
• Continuous monitoring of cells over extended periods of time using automated imaging systems to analyze cellular processes such as adhesion, proliferation, invasion, migration and cell to cell interaction

Assay summary