What is live cell imaging?
Live cell imaging is the study of cellular structure and function in living cells via microscopy. It enables the visualization and quantitation of dynamic cellular processes in real time. The ability to study cellular and subcellular structure, function, and organization in living systems aids in the development of assays that are more biologically relevant and that can better predict the human response to new drug candidates. Live cell imaging encompasses a broad range of topics and biological applications—whether it is performing long-term kinetic assays or fluorescently labeling live cells.
Time-lapse video of fibroblast cell division. Time-lapse imaging was performed for a total time of 18 hours. Fibroblasts stably expressed mCherry-H2B (red, nuclei) and GFP-β-tubulin (green, tubulin)
Benefits of live cell imaging
Live cell imaging is a fundamental research tool in cell biology labs and in a wide variety of industries, and it has led to the discovery of drug targets and candidates as well as the molecular mechanisms involved in diseases. Critical for these researchers, is the ability to develop assays that can best mimic the in vivo nature of cells. Benefits of imaging live cells include:
- Monitoring live cells over time provides more than a snapshot of what is occurring in the cells while allowing for the visualization of transient events that may be missed in end-point assays and revealing optimal time-points for end-point assays.
- Tight regulation of environmental conditions ensures that the cellular structures and processes being studied are in their native physiological state
- Imaging live cells in their natural state minimizes artifacts that can arise from other imaging techniques, such as cell fixation and immunostaining.
- The localization and transport of cellular biomolecules along with the progression of multiple pathways can be investigated simultaneously in real-time.
- Acquiring images laterally, axially, and temporally enables the generation of 4D images and data
- Live cell imaging of molecular dynamics generates qualitative and quantitative data that can’t be gained from other biochemical methods.
Cellular imaging and analysis workflow
Protocols for culturing, plating, and maintaining live cells will vary between different cell models however, the steps below outlines a generalized workflow for live cell imaging assays.
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Plate cells into labware of choice (chamber slides, dishes, microplates)
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Treat cells – Perform cell treatments with desired compounds, RNAi, etc. if part of the assay workflow.
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Stain for markers – If required, label cells with the desired fluorophores (fluorescent dyes, fluorescent protein – peptide fusions, etc.
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Environmental controls – If the acquisition will take a long time or if the assay is a live cell time-lapse experiment (continuous or discontinuous), then enable full environmental control including gas, temperature, and humidity.
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Acquire cell image – Place the plate into the automated imaging system and start acquiring your live cell images
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Analyze cell image – Use cellular imaging analysis software to run quantitative analysis of live cell images
Live cell imaging system
Sophisticated live cell imaging instruments provide the ability to regulate environmental conditions to maintain the health and viability of cells while monitoring molecular and cellular dynamics from the single cell to organismal level. From the study of fast-kinetic events to running long-term, time-lapse assays, automated live cell imaging provides the direct observation of dynamic biological processes and the ability to generate a wealth of cellular information
- Label-free, brightfield imaging to multi-color fluorescence imaging capabilities provide the flexibility necessary for the multitude of live cell imaging applications
- High-acquisition speeds allow for the examination of fast-kinetic events, such as calcium oscillation in cardiomyocytes.
- Robust focusing capabilities of automated live cell imaging instruments ensure the maintenance of optimum focus through the entirety of the acquisition, which is crucial for generating accurate live cell imaging data.
Applications and assays
Advancements in automated microscopes with integrated environmental control chambers as well as in fluorescent protein and synthetic fluorophore technology have expanded the capabilities of researchers investigating biological processes in living cells.
Our systems for high-content imaging and analysis provide flexible scalability making it easy to evolve your system alongside your research. They feature options and modules to address your specific research needs including objectives, filters, imaging modes, and environmental control. Here we have a collection of applications and assays that utilize these key features for your live cell imaging research:
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3D Live Cell Assays
The integration of three-dimensional (3D) assay models is becoming more widespread to drive translational biology. This has led to significant progress in the development of higher complexity and biologically relevant 3D cell models to better mimic in vivo environments and responses to drug treatments. Recently, simple, automated workflows, suitable for high-content imaging have become popular. Acquiring measurements from these 3D structures involve acquiring images from different depths (z-planes) and analyzing them in 3D or collapsing the images into a single 2D stack before analysis.
- Acquire data 10X faster from 3D spheroids cultured using Symphony® and VersaGel®
- High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
- 3D analysis and morphometric characterization of compound effects on cancer spheroid cultures
- High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
- 3D Cell Models
3D Live Cell Assays
The integration of three-dimensional (3D) assay models is becoming more widespread to drive translational biology. This has led to significant progress in the development of higher complexity and biologically relevant 3D cell models to better mimic in vivo environments and responses to drug treatments. Recently, simple, automated workflows, suitable for high-content imaging have become popular. Acquiring measurements from these 3D structures involve acquiring images from different depths (z-planes) and analyzing them in 3D or collapsing the images into a single 2D stack before analysis.
- Acquire data 10X faster from 3D spheroids cultured using Symphony® and VersaGel®
- High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
- 3D analysis and morphometric characterization of compound effects on cancer spheroid cultures
- High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
- 3D Cell Models
Cell Migration Assays
The movement or migration of cells is often measured in vitro to elucidate the mechanisms of various physiological activities such as wound healing or cancer cell metastasis. Cell migration assays may be conducted in a controlled environment using live cell time lapse imaging. A "wound" in a confluent monolayer of cells growing in a microplate is created, either by manually creating a scratch or by utilizing special microplates that provide a uniform and reproducible cell-free zone. Monitor cell proliferation, wound healing, migration, and spreading using transmitted light or live cell-compatible fluorescence. These medium to high throughput assays may be used to compare the migration between cells treated with either inhibitory or stimulatory compounds.
Cell Migration Assays
The movement or migration of cells is often measured in vitro to elucidate the mechanisms of various physiological activities such as wound healing or cancer cell metastasis. Cell migration assays may be conducted in a controlled environment using live cell time lapse imaging. A "wound" in a confluent monolayer of cells growing in a microplate is created, either by manually creating a scratch or by utilizing special microplates that provide a uniform and reproducible cell-free zone. Monitor cell proliferation, wound healing, migration, and spreading using transmitted light or live cell-compatible fluorescence. These medium to high throughput assays may be used to compare the migration between cells treated with either inhibitory or stimulatory compounds.
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Cytotoxicity Assays
Screening for off-target or toxic effects is very important during the development of new drugs and for the extension of the therapeutic potential of existing molecules. ImageXpress systems are fully integrated hardware and software platforms for automated acquisition and analysis of images for high-throughput cell-based cytotoxicity testing. Configured with optional environmental control, living cell responses or kinetic reactions can be monitored in real time for several days.
- Increase accuracy and efficiency of cytotoxicity assessment using the EarlyTox Live/Dead Assay Kit and high-content imaging
- Assaying cardiotoxicity with ImageXpress high-content screening systems
- Apoptosis detection using EarlyTox Caspase-3/7-D NucView 488 Assay Kit on ImageXpress Micro systems
- Multiplexed high content hepatotoxicity assays using iPSC-derived hepatocytes
- High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
- Cytotoxicity assessment using automated cell imaging and live/dead assays
Cytotoxicity Assays
Screening for off-target or toxic effects is very important during the development of new drugs and for the extension of the therapeutic potential of existing molecules. ImageXpress systems are fully integrated hardware and software platforms for automated acquisition and analysis of images for high-throughput cell-based cytotoxicity testing. Configured with optional environmental control, living cell responses or kinetic reactions can be monitored in real time for several days.
- Increase accuracy and efficiency of cytotoxicity assessment using the EarlyTox Live/Dead Assay Kit and high-content imaging
- Assaying cardiotoxicity with ImageXpress high-content screening systems
- Apoptosis detection using EarlyTox Caspase-3/7-D NucView 488 Assay Kit on ImageXpress Micro systems
- Multiplexed high content hepatotoxicity assays using iPSC-derived hepatocytes
- High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
- Cytotoxicity assessment using automated cell imaging and live/dead assays
eBook: Cellular Imaging Insights
Elucidating complex cellular pathways within 2D and 3D cellular structures in response to chemical stimulation or stress is the goal of many researchers. The capacity to which scientists have access to and can adopt emerging cellular imaging and analysis technology fuels their level of understanding.
eBook: Cellular Imaging Insights
Elucidating complex cellular pathways within 2D and 3D cellular structures in response to chemical stimulation or stress is the goal of many researchers. The capacity to which scientists have access to and can adopt emerging cellular imaging and analysis technology fuels their level of understanding.
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Label-Free Live Cell Assays
Label-free imaging and analysis is a requirement for a multitude of live cell biological applications that monitor cell number, confluency, cytotoxicity, development, growth, or proliferation. High contrast brightfield and phase contrast imaging along with robust analysis capabilities provide precise segmentation for the quantification of cells and the assessment of a variety of cellular responses and morphologies.
Label-Free Live Cell Assays
Label-free imaging and analysis is a requirement for a multitude of live cell biological applications that monitor cell number, confluency, cytotoxicity, development, growth, or proliferation. High contrast brightfield and phase contrast imaging along with robust analysis capabilities provide precise segmentation for the quantification of cells and the assessment of a variety of cellular responses and morphologies.
Perform Functional Assays with Living Cells
It is critical for drug screening programs to gain a global view of the cytotoxic effects of potential drug candidates. In combination with traditional cytotoxicity assays, functional assays provide insight into the effects of drugs and compounds on normal cellular function. Sensitive functional assays like calcium flux assays, which utilize a fluorescent dye to monitor changes in intracellular calcium, aid in understanding the full scope of cytotoxicity. Calcium flux assays have been utilized to investigate the effects of compounds on cardiomyocyte pacing and neuron electrophysiology. Maintaining physiological environmental conditions and being able to perform fast kinetic imaging is ideal for running functional assays.
- High-throughput screening of single-cell contractile force using FLECS Technology
- Explore cardiac function by measuring calcium oscillation or contraction patterns
- Multiplexed automated imaging assay for cardiotoxic compounds using ImageXpress Pico system
- Assessment of drug effects on cardiomyocyte physiology using human iPSC-derived cardiac spheroids
Perform Functional Assays with Living Cells
It is critical for drug screening programs to gain a global view of the cytotoxic effects of potential drug candidates. In combination with traditional cytotoxicity assays, functional assays provide insight into the effects of drugs and compounds on normal cellular function. Sensitive functional assays like calcium flux assays, which utilize a fluorescent dye to monitor changes in intracellular calcium, aid in understanding the full scope of cytotoxicity. Calcium flux assays have been utilized to investigate the effects of compounds on cardiomyocyte pacing and neuron electrophysiology. Maintaining physiological environmental conditions and being able to perform fast kinetic imaging is ideal for running functional assays.
- High-throughput screening of single-cell contractile force using FLECS Technology
- Explore cardiac function by measuring calcium oscillation or contraction patterns
- Multiplexed automated imaging assay for cardiotoxic compounds using ImageXpress Pico system
- Assessment of drug effects on cardiomyocyte physiology using human iPSC-derived cardiac spheroids
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Subcellular Studies
Live cell imaging permits investigation into a variety of cellular processes involving subcellular compartments and molecules. This includes the monitoring of kinetic processes in real time, such as autophagy, cell signaling, protein trafficking, receptor internalization, endocytosis, and translocation to name a few. Advancements in automated imaging systems as well as in fluorescent protein and synthetic fluorophore technology has expanded the capabilities of researchers to study the dynamic function and structure of organelles and proteins.
Subcellular Studies
Live cell imaging permits investigation into a variety of cellular processes involving subcellular compartments and molecules. This includes the monitoring of kinetic processes in real time, such as autophagy, cell signaling, protein trafficking, receptor internalization, endocytosis, and translocation to name a few. Advancements in automated imaging systems as well as in fluorescent protein and synthetic fluorophore technology has expanded the capabilities of researchers to study the dynamic function and structure of organelles and proteins.
Time-Lapse Imaging
Many live cell imaging applications require maintenance of native cellular environmental conditions and the ability to perform time-lapse imaging. This includes the ability to regulate temperature, pH, gas (O2, CO2), and humidity. Being able to maintain these critical components allows for the preservation of cell health during long-term, multi-day assays. The ImageXpress suite of imaging systems provide the seamless integration of environmental control, robust imaging, and powerful analysis enabling the acquisition and visualization of kinetic images and data.
Time-Lapse Imaging
Many live cell imaging applications require maintenance of native cellular environmental conditions and the ability to perform time-lapse imaging. This includes the ability to regulate temperature, pH, gas (O2, CO2), and humidity. Being able to maintain these critical components allows for the preservation of cell health during long-term, multi-day assays. The ImageXpress suite of imaging systems provide the seamless integration of environmental control, robust imaging, and powerful analysis enabling the acquisition and visualization of kinetic images and data.
Live Cell Imaging and Analysis Workflow
The described live cell imaging workflow is a generalized example of a continuous long-term time-lapse live cell assay. This workflow breaks down the steps from cell plating to image analysis and highlights the materials and instrumentation necessary to run a live cell assay including an automated high-content imaging system and cellular imaging and analysis software.
PLATE CELLS
Plate adherent or suspension cells into the labware of choice including culture dishes, chamber slides, and microplates. Incubate the cells at normal culture conditions (i.e. 37°C, 5% CO2). For adherent cells, incubating the plated cells overnight will allow ample time for cell attachment and growth.
96- or 384- Well Plates
Most cell cultures for high-content, high-throughput screening are run using 96- or 384-well microplates.
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TREAT WITH COMPOUNDS
If necessary for the protocol, treat the cells with compounds of interest.
Compounds can be added to cells for minutes up to several days depending on the mechanism of action of the compounds and the biological re-sponse being investigated. For example, apoptosis or receptor internalization assays generally require shorter incubation periods while multiparametric cytotoxicity assays require longer periods. Longer compound treatments may require the replacement of compounds during incubation
STAIN FOR MARKERS
Live cells can be stained with a variety of fluorophores or transfected/transduced with constructs containing fluorescent protein – peptide fusions. Manufacturer instructions should be followed when staining. Stain-ing isn’t necessary for all live cell applications and label free imaging and analysis allows for the tracking and monitoring of cells in brightfield only.
Assay Kits
Easy-to-use, robust assay kits for life science research, drug discovery and development, and bioassays. Our assay kits are optimized for use on our instruments. Screen more compounds earlier in drug discovery and enable characterization of a full concentra-tion-response profile of test compounds.
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ImageXpress High-content Imaging Portfolio
Our systems for high-content imaging and analysis provide flexible scalability making it easy to evolve your system alongside your research. They feature options and modules to address your specific research including objectives, filters, imaging modes, and envi-ronmental conditions.
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CONFIGURE ENVIRONMENTAL CONTROL SETTINGS
After the addition of stains and compounds, place the plate into the appropriate imaging instrument integrated with environmental control capabilities such asl gases (CO2, O2), temperature, and humidity.
For long-term time-lapse assays it is essential to maintain a natural physiological environment to ensure the health of living cells and to prevent focus drift throughout the time-lapse acquisition.
ACQUIRE LIVE CELL IMAGE
For continuous time-lapse acquisitions, configure the total length of time for the time series and the imaging intervals, i.e. image every hour for 24 hours.
Discontinuous time-lapse acquisitions can be per-formed, where the plate is taken to and from the incubator and imaged at different time points.
Automated Cell Imaging System
The ImageXpress® Pico Automated Cell Imaging System is more than a digital microscope, combining high-resolution imaging with powerful analysis. Whether running fluorescence imaging or brightfield assays, the automated imager features comprehensive preconfigured protocols for cell-based assays to shorten the learning curve, so you can start running experiments quickly.
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Cellular Imaging Acquisition and Analysis Software
The CellReporterXpress Automated Image Acquisition and Analysis Software works with the ImageXpress® Pico systems. It has a clean, easy-to-learn interface for performing quantitative analysis on images acquired from automated microscopy. The software enables distributed analysis of images for increased throughput and is ideal for scaling microscopy imaging with slides or microplates.
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ANALYZE LIVE CELL IMAGE
Analyze the images with cellular imaging analysis software to generate multiparametric readouts for the biological responses that are being studied. Kinetic data can be generated and reviewed for time-lapse acquisitions.
Resources for Live Cell Imaging
Application Note
Monitor multiple stages of apoptosis with live cell kinetic imaging
Monitor multiple stages of apoptosis with live cell kinetic imaging
The study of apoptosis is a critical aspect of drug discovery and development. Additionally, studying the relationship between apoptosis and other factors, such as oxidative stress, is…
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Application Note
Monitor cell proliferation and cell cycle in real time
Monitor cell proliferation and cell cycle in real time
There is an increased need to expand the variety and complexity of cell-based assays for biological research and drug discovery. Live-cell assays allow monitoring of cell responses in real…
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Application Note
Explore cardiac function by measuring calcium oscillation or contraction patterns
Explore cardiac function by measuring calcium oscillation or contraction patterns
Understanding the impact of drugs on cardiac system early in the development process is critical to improve the safety of drugs getting to market. Cardiotoxicity is believed to be one of…
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Application Note
Count cells with or without fluorescent labels using automated imaging
Count cells with or without fluorescent labels using automated imaging
The ability to accurately quantitate cell number in multi-well microplates enables a multitude of biological applications that study cell health or proliferation.
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Application Note
High-throughput screening of single-cell contractile force using FLECS Technology
High-throughput screening of single-cell contractile force using FLECS Technology
The ability of mammalian cells to generate mechanical forces—to push, pull, or squeeze—is an intrinsic capability that is used by cells both individually and together as tissue to perform…
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Application Note
Measure cell migration using discontinuous time-lapse imaging of live cells
Measure cell migration using discontinuous time-lapse imaging of live cells
Cell migration is an essential process required for many biological events including embryonic development, wound healing, cancer metastasis, and immunological responses.
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Application Note
Multiplexed automated imaging assay for cardiotoxic compounds using the ImageXpress Pico system
Multiplexed automated imaging assay for cardiotoxic compounds using the ImageXpress Pico system
Develop functional and morphological readouts for testing effects of different compounds in multi-parametric assays using iPSC-derived cardiomyocytes
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Application Note
Acquire data 10X faster from 3D spheroids cultured using Symphony® and VersaGel®
Acquire data 10X faster from 3D spheroids cultured using Symphony® and VersaGel®
Most in vitro cell culture work is performed in two-dimensions (2D), where cells grow on a flat surface. However, culturing cells in 2D to screen drugs provides a limited picture of what…
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Application Note
Detection of autophagy using automated imaging
Detection of autophagy using automated imaging
Detect and quantify compound effects on the process of autophagy on the ImageXpress Pico system. The PC12 human neuroblastoma cell line was used as a model for assay development.
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Application Note
Evaluating cell cycle inhibitors using a live cell assay
Evaluating cell cycle inhibitors using a live cell assay
Monitoring treatment effects on the cell cycle is relevant to progressing oncology research and drug discovery. High-content screening assays using live cells have been developed to enable…
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Application Note
Evaluate cell migration with FluoroBlok inserts on the SpectraMax MiniMax cytometer
Evaluate cell migration with FluoroBlok inserts on the SpectraMax MiniMax cytometer
Cell migration, broadly defined as the movement of cells from one location to another, is important in diverse processes including embryonic development and wound healing. It is also a key…
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Application Note
Cytotoxicity assessment using automated cell imaging and live/dead assays
Cytotoxicity assessment using automated cell imaging and live/dead assays
Live/dead assays are utilized in a wide variety of research applications including investigation of cytotoxic effects of various compounds, treatments, or changes in gene expression.
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Application Note
Measuring mitochondrial shape and distribution using the ImageXpress Micro High-Content Imaging System
Measuring mitochondrial shape and distribution using the ImageXpress Micro High-Content Imaging System
Using high-content imaging assays allow cells cultured and treated in microplates to be imaged alive without wash steps for real-time studies of cells in their most natural state, although…
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Application Note
Assessment of drug effects on cardiomyocyte physiology using human iPSC-derived cardiac spheroids
Assessment of drug effects on cardiomyocyte physiology using human iPSC-derived cardiac spheroids
Cell models are becoming more complex in order to better mimic in vivo microenvironments and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest…
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Application Note
High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
Establishment of physiologically-relevant in vitro models is crucial to further understanding of the mechanisms of neurological diseases as well as targeted drug development. While iPSC-…
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Application Note
3D analysis and morphometric characterization of compound effects on cancer spheroid cultures
3D analysis and morphometric characterization of compound effects on cancer spheroid cultures
Cellular transformation/tumorigenicity assays using cultures of cells in semi-solid media (soft agar or Matrigel) has been well established for cancer research1,2,5. The assay requires…
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Application Note
High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
There is increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in…
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Application Note
High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
In recent years, there has been significant progress in development of in vitro aggregates of tumor cells for use as models for in vivo tissue environments. When seeded into a well of a…
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Application Note
Alternatives to DAPI staining: imaging and counting live cells
Alternatives to DAPI staining: imaging and counting live cells
DAPI (4’, 6-diamidino-2-phenylindole) is a fluorescent dye often used to stain nuclear DNA. It is employed in imaging experiments such as fluorescent microscopy, chromosome spreads, FACS,…
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Application Note
Increase accuracy and efficiency of cytotoxicity assessment using the EarlyTox Live/Dead Assay Kit and high-content imaging
Increase accuracy and efficiency of cytotoxicity assessment using the EarlyTox Live/Dead Assay Kit and high-content imaging
Cell viability assays are utilized in a wide variety of research areas ranging from examining the mechanisms of cell death and cell proliferation to evaluating the effects of cytotoxic…
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Application Note
Live cell kinetics assay utilizing the ImageXpress Micro System
Live cell kinetics assay utilizing the ImageXpress Micro System
The ImageXpress® Micro Confocal/ XLS Widefield High-Content Imaging System from Molecular Devices includes optional Fluidics and Environmental Control modules, enabling single-channel…
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Application Note
Assaying cardiotoxicity with ImageXpress high-content screening systems
Assaying cardiotoxicity with ImageXpress high-content screening systems
The discovery and development of novel drugs is a difficult and expensive process due to lack of tools and techniques to accurately reproduce the human physiological response in the…
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Application Note
Visualize subcellular vesicles to quantitate autophagy in neuronal cells
Visualize subcellular vesicles to quantitate autophagy in neuronal cells
Autophagy is an intracellular catabolic process that sequesters and degrades proteins and organelles that have either been recognized as faulty or are simply no longer needed by the cell.…
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Application Note
Apoptosis detection using EarlyTox Caspase-3/7-D NucView 488 Assay Kit on ImageXpress Micro systems
Apoptosis detection using EarlyTox Caspase-3/7-D NucView 488 Assay Kit on ImageXpress Micro systems
Apoptosis is an important mechanism signaling programmed death of cells in normal processes such as embryonic development, as well as in diseases including cancer and neurodegenerative…
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Application Note
Intelligent time-lapse imaging to enhance live cell assay development
Intelligent time-lapse imaging to enhance live cell assay development
The ability to monitor responses in living cells over a specific period of time offers cell biologists several key advantages for assay development. For routine cellbased screening, time-…
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Application Note
Multiplexed high-content hepatotoxicity assays using iPSC-derived hepatocytes
Multiplexed high-content hepatotoxicity assays using iPSC-derived hepatocytes
Drug-induced hepatotoxicity is an important cause for liver injury and acute liver failure. Thus highly predictive assays for safety and efficacy testing are crucial for improving drug…