Operetta CLS High-Content Analysis System

Extracellular signal-regulated kinase (ERK) is a key component in the regulation of embryogenesis, cell differentiation, cell proliferation, and cell death. The ERK signaling pathway is altered in various cancer types and is frequently investigated as a target for therapeutic intervention. This application note describes how a live cell FRET assay to study ERK signaling was performed on the Operetta CLS™ high-content analysis system. The optimized design of the FRET-based biosensor, the high-quality imaging of the Operetta CLS system and the easy-to-use image analysis tools of the Harmony® software contribute to the robustness of the high-content assay.

In this application note, we describe a high-content screening application for analyzing the migration of non-small cell lung cancer cells in a live cell assay. Using the Operetta® high-content imaging system and digital phase contrast imaging, we tracked migrating cancer cells using automated single cell tracking in the Harmony® high-content imaging and analysis software.

The promise of high-content screening is the acceleration of discovery by extracting as much relevant information as possible from cells. Nevertheless, a large percentage of high-content screens analyze only a small number of image-based properties. As a result, valuable information from precious cells and disease models is not utilized. As nearly all screening approaches require a nuclear counterstain such as Hoechst to facilitate segmentation, phenotypic profiling of the nuclei can offer new and additional perspectives on assays at no extra cost.

In this application note, we demonstrate an efficient cell-based workflow for the assessment of EGF treatment effects in a cellular model of human skin cancer.

Treatment effects on several intracellular signaling pathways were examined using PerkinElmer’s homogeneous, no-wash AlphaLISA^® SureFire^® Ultra assays. To determine concurrent time-dependent effects of different EGF concentrations on cellular health and proliferation, ATP concentrations were assessed with ATPlite™ 1step luminescence assay and cultures were fluorescently labeled, imaged and analyzed using the Operetta CLS™ high-content analysis system.

Learn how a phenotypic screening assay to study time-dependent effects of endothelin-1-induced hypertrophy was set up using human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Learn how: The Opera Phenix system has been applied in the field of neurodegenerative diseases. In this assay, the Opera Phenix system is 4 times faster than the previous Opera® system. Primary neuron morphology is analyzed in a straightforward approach using Harmony software. Careful assay optimization can increase throughput, and minimize the data burden, without compromising assay performance.

Whether you’re familiar with high-content screening and are looking to exploit the increased physiological relevance of complex 3D cell models, or you want to take your analysis of 3D cell models to the next level, migrating from simple plate-reader assays to a high-content approach, you’ll need the right tools and strategies to overcome the challenges these models present.

Live-cell imaging, the study of living cells using microscopy, has become a requisite technology in many fields of biomedical research, such as cell biology, developmental biology and cancer research. Also, in drug discovery, researchers adopt live-cell imaging as they look for a more detailed understanding of cellular behavior.

In our article, learn about:

• The 3 different categories of live-cell assays
• 5 key benefits of live-cell imaging assays
• Examples of key live-cell assays, e.g. calcium flux, immune-cell killing, single-cell tracking

Download our brochure to learn how our solutions help you to grow, detect, and analyze 3D cells.

One of the greatest challenges in multiple sclerosis (MS) therapy is the halting or reversal of the failure of remyelination in the brain in order to reverse disabilities in MS patients. This case study highlights the recent work of Dr. Paul Tesar and colleagues at the Case Western Reserve University School of Medicine, which could potentially lead to such novel treatments, as it aims to control the function of stem cells in the body and thereby to help the body repair itself.

Antimicrobial resistance (AMR) is one of today’s major global public health challenges. Recently, there has been renewed investment in the discovery of novel antimicrobials to urgently address the growing number of drug-resistant infections. This case study describes how Professor Gordon Dougan and his team at the Cambridge Biomedical Research Centre are using high-content analysis to phenotype individual bacteria within a population to investigate adaptive mechanisms of antimicrobial resistance, and to screen for novel alternatives to existing antimicrobials.

Download this booklet from The Scientist and PerkinElmer to learn about how the third dimension affects cell behavior, the similarities and differences between 2-D and 3-D culture, common 3-D culture models, and how to image and analyze 3-D culture models.

While 3D cell culture provides unprecedented opportunities for both increased physiological relevance and analysis using a high-content approach, it is also more complex than traditional 2D cell culture. This booklet, from Biocompare and PerkinElmer, will unravel some of the complexities often encountered when using 3D cell models for drug discovery and provide insights and solutions that will streamline workflows and facilitate the development of effective therapeutics. Topics covered include: Reagents and instruments for growing, detecting, and analyzing 3D cell models; 3D culture methodologies; the value of high-content screening with 3D cell models and how to improve image acquisition and image analysis with high-content assays.

Infectious diseases remain a major burden to human health. The increased globalization of modern society that facilitates the spread of infectious diseases, and phenomena such as anti-microbial resistance, underscore the importance of the development of new preventative and therapeutic approaches.

Download this booklet to learn how high-content imaging and analysis enable high-throughput functional and phenotypic assays that can be adapted to a wide range of pathogens; read a series of Featured Publication Notes describing the contribution of high-content analysis in the study of diseases such as ebola, zika, tuberculosis, listeria and malaria and find examples of studies in which a high-content approach has been used in parasitic, viral and bacterial disease research.

Please download the pdf and view in Adobe Reader or Acrobat for optimum performance.

Live-cell imaging has evolved from allowing observation of large-scale changes to capturing subtle changes in dynamic cellular processes. Today, live-cell imaging coupled with high-content analysis enables researchers to extract quantitative data in real-time, facilitating new insights in basic life science research and drug discovery. Modern live-cell imaging systems can capture rapid cellular events, track cell movement, monitor protein signaling, screen cell health, and much more.

Download the eBook from The Scientist and PerkinElmer to discover:

• Early attempts at live-cell imaging
• Key challenges and considerations for live-cell imaging
• A guide for choosing the appropriate imaging system and optimizing cell health
• Advances in live-cell imaging technology

Whether you’re familiar with high-content screening, or a newcomer, you’ll need the right tools and strategies to overcome the challenges of using complex 3D cell models in such an assay. For example, growing consistent, reproducible 3D cultures can be problematic and imaging large, thick cell samples can be challenging, while managing the huge volumes of data generated is perhaps the most demanding aspect of all. In this article, we provide our top tips for running a successful high-content screening assay using a 3D cell model. Learn how you can: Generate uniform 3D cell models, Get the best quality images, Minimize imaging time and volume of data, Get deeper insights from your 3D cell model and Avoid unnecessary data transfer steps.

High-content assays using 3D objects such as cysts or organoids can be challenging from the perspectives of both image acquisition and image analysis. In this technical note we describe how to image and analyze epithelial Madin-Darby canine kidney (MDCK) cysts in 3D on the Operetta CLS^™ high-content analysis system.

We address:

• How to reduce the loss of image resolution as you image deeper into the sample
• How to enable 3D segmentation and analysis for your 3D high-content assays
• How to increase throughput for 3D imaging through shorter exposure times
• How to eliminate long and tedious data transfer steps

Multicellular 3D “oids” (tumoroids, spheroids, organoids) have the potential to better predict the effects of drug candidates during preclinical screening. However, compared to 2D cell monolayers, assays using 3D model systems are more challenging.

In this technical note we describe how to image and analyze solid spheroids in 3D using the Opera Phenix^™and Operetta^™CLS high-content screening systems and Harmony^® 4.8 imaging and analysis software.

We address:

In drug discovery programs, multicellular spheroids have emerged as powerful tools to bridge the gap between in vitro cell culture models and in vivo tissues. However, one of the greatest challenges in higher throughput 3D imaging is the acquisition of images of solid spheroids, owing to the reduced light penetration.

One solution is to use optical clearing techniques, which can enhance the imaging depth in spheroids by removing lipid and protein molecules.

In this technical note, we compare different optical clearing strategies for 3D spheroids and quantify the clearing effectiveness and alterations in spheroid morphology, and demonstrate how to increase imaging depth in 3D spheroids by a factor of four.

Live cell imaging has gained importance within drug discovery over recent times, as researchers look for more meaningful insights into cellular behavior and function. However, setting up live cell experiments can be challenging, as temperature, CO₂ and evaporation need to be controlled to ensure optimal cell growth conditions. In this technical note, we demonstrate:

Download our technical note to find out how you can overcome some of the challenges associated with long-term live cell imaging. Learn how you can perform successful five-day live cell imaging on Operetta CLS™ and Opera Phenix™ high-content systems, avoid phototoxicity with gentle digital phase contrast imaging, and analyze cell growth and morphology on a single cell level without fluorescence staining.

Automated image acquisition and analysis of tissue sections can be challenging owing to the inherent height variations throughout each specimen, and this can hinder their use in screening applications.

In this technical note we show how PreciScan^™ intelligent image acquisition enables generation of accurate high-resolution images, overcoming issues caused by factors such as height variations in the specimen or varying positions of the tissue sections on the slides, while also significantly reducing the data volume and acquisition time.

Spinning disk confocal microscopy is a common tool for live cell microscopy and reduces background fluorescence from out of focus planes. However, for 3D imaging, confocal image quality can be limited by a phenomenon known as ‘pinhole crosstalk’ which can lead to blurred images in conventional spinning disk systems. The confocal scanning unit of the Opera Phenix^™ High-Content Screening System provides an increased distance between pinholes to reduce the pinhole crosstalk, leading to improved 3D image quality. Learn more in our Technical Note.

The key parameters in high-content imaging - speed, sensitivity (or intensity) and resolution - cannot be optimized independently as altering one of them influences the others. For example, increasing the resolution by choosing a higher magnification requires imaging of more fields to maintain the same cell number – at the expense of speed; or increasing the fluorescence intensity can be achieved by increasing the exposure time – again at the expense of speed. Nevertheless, there is a way to overcome some of these obstacles and here, in this Technical Note, we explain why the choice of the objective lens is critical.

Researchers are increasingly looking to 3D cell cultures, microtissues, and organoids to bridge the gap between 2D cell cultures and in vivo animal models. This whitepaper documents a streamlined procedure for getting the most information, as quickly as possible, using solutions from PerkinElmer.

Today's drug discovery strategies require candidate compounds to fail early and cheaply in the discovery stage, rather than late and expensively in the clinical phase. Testing compounds early in physiologically relevant model systems and leveraging rich information from image-based screens are ways of focusing on those compounds that give rise to the right phenotypic changes without undesirable effects on the system.

Learn:

1. Why you should consider phenotypic vs. target based drug discovery strategies
2. Challenges you should be aware of for phenotypic assays
3. How to maximize the return on your phenotypic assays