Seahorse XF Pro Analyzer

In this application note, Agilent Technologies presents the evaluation of 1,3-diphenylguanidine (DPG) and five of its chlorinated derivatives’ effects on cell heath and bioenergetics using the Agilent xCELLigence RTCA eSight and Agilent Seahorse XF96 extracellular flux analyzer. 1-(2,4-dichlorophenyl)-3-phenylguanidine (CC15) and 1-(4-chlorophenyl)-3-(2,4-dichlorophenyl) guanidine (CC11) were the least and most cytotoxic compounds, respectively, except for DPG, which was the most cytotoxic. Real time cell analysis (RTCA) results were similar to traditional alamarBlue cell viability assay results. The Seahorse XF96 analyzer results indicated that, except for 1,3-bis-(2-chlorophenyl) guanidine (CC04), all of the DPG compounds tested had adverse effects on cell bioenergetics. While the compounds’ limited effects on mitochondrial basal respiration and ATP production might be short lived, the significant effect on proton leakage suggested irreversible mitochondrial damage.

Decreased oxygen levels, or hypoxia, and hypoxic-mediated signaling play an important role in the pathology of diseases including stroke, ischemic heart disease, renal fibrosis, and cancer. In this application note, learn more about the metabolic differences in MCF-7 human breast cancer cells cultured and assayed at either ambient or 5 % oxygen levels, by measuring mitochondrial respiration and glycolytic activity using the Agilent Seahorse XF Analyzer and XF Hypoxia Rate Calculator.

The Agilent Seahorse XF T Cell Metabolic Profiling kit is a robust solution for the complete real-time assessment of T cell metabolism. In this application note, learn more about using the Agilent kit to assess the impact of the medium composition during T cell expansion following the XF T Cell Persistence Assay workflow. This workflow allows for the evaluation of glycolysis and mitochondrial activity and capacity simultaneously, providing a comprehensive metabolic profile of T cells that can be incorporated into monitoring and improving the design and development of T cell therapies.

Pathogenic viruses have different strategies to ensure ideal environments for their replication and spread, and have evolved to alter numerous host cell pathways. A greater understanding of these metabolic alterations required for viral replication may lead to innovative therapeutic tactics through targeted modification of specific mitochondrial functions or pathways. In this application note, learn about how the Agilent Seahorse XF technology delivers a real-time view of the fundamental processes of energy metabolism in live cells. Understand how this capability enables convenient interrogation of the metabolic implications of pathogen infection on host cells, including those specific to mitochondrial function, offering new insights on key host-pathogen interactions.

Learn more about the Agilent Seahorse XF Mito Tox Assay, a customized XF solution that allows the detection and quantitative assessment of compounds eliciting mitochondrial toxicity. This application note provides proof of concept and examples for compound screening, dose-response experiments using the XF Mito Tox assay, and interpretation of resulting data using the Mito Tox Index (MTI). It also presents a discussion of strategic assay design, performance metrics, and specific cases when using the XF Mito Tox Assay.

In this application note, learn more about the use of the Agilent XF T cell metabolic profiling kit. This new product uses an improved uncoupler (BAM15) for more consistent and accurate measurements of T cell bioenergetic capacity with less concentration optimization required than FCCP. It can be used to assess the impact of the medium composition during T cell expansion following the Agilent XF T Cell Persistence Assay workflow. This workflow allows for the evaluation of glycolysis and mitochondrial activity and capacity simultaneously, providing a comprehensive metabolic profile of T cells that can be incorporated into monitoring and improving the design and development of T cell therapies.

The regulation of cellular bioenergetics is recognized as a key driver of cancer cell proliferation. A new workflow for rapid functional screening of agents modulating cancer cell metabolism is applied using the Agilent Seahorse XF Real-Time ATP rate assay combined with up-to-date data analysis software. This application note introduces how informative the XF Real-Time ATP rate assay data is in selecting metabolic modulators and evaluating drug potency. A workflow of anticancer drug screening with a kinase inhibitor library is also demonstrated. This workflow can be used to identify drug compounds targeting cancer cell metabolism or druggable targets that impact mitochondrial and glycolytic metabolism.

Mitochondria are critical regulators of normal cell function and health; consequently, their dysfunction plays a central role in many diseases. Drug-induced mitochondrial dysfunction has been linked to compound attrition, organ toxicity, black-box warnings, and market withdrawals, therefore it is important to identify mitochondrial toxicity during the early stages of the drug discovery process. Download this infographic to explore the impact of mitochondrial toxicity and learn of the benefits of novel in-vitro strategies for drug safety.

Join Professor Mercedes Rincon, University of Colorado Anschutz School of Medicine, as she discusses the groundbreaking advancements in adoptive T cell therapy, particularly chimeric antigen receptor (CAR) T cell therapy. While CAR-T therapy has achieved impressive complete remission rates in 60–90% of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (r/r-B-ALL), challenges such as treatment failures and relapses remain significant obstacles.

Metabolism plays a critical role in T cell function, shaping immune responses. While glycolysis is essential for T cell expansion, it can restrict the self-renewal capacity of CAR-T cells during manufacturing. Mitochondrial respiration is vital for their survival and effectiveness. Recent findings have highlighted MCJ, a protein that negatively regulates mitochondrial function in CD8 cells. Loss of MCJ enhances mitochondrial respiration, cytokine secretion, and anti-tumor activity. Targeting MCJ to boost mitochondrial metabolism presents a promising strategy to enhance the efficacy of CAR-T cells and advance adoptive T cell therapies.

Key learning objectives

• Understand the current landscape of adoptive T-cell therapy
• Explain the role of metabolism in T cell function, highlighting the significance of glycolysis and mitochondrial respiration in enhancing CAR-T cell efficacy
• Identify the potential of MCJ as a therapeutic target to improve mitochondrial metabolism, cytokine secretion, and overall effectiveness of CAR-T cell therapies

Who should attend?

• Researchers and scientists specializing in CAR T-cell therapy, immunotherapy development, cell metabolism, glycolysis, and mitochondrial respiration.

Certificate of attendance
All webinar participants can request a certificate of attendance, including a learning outcomes summary, for continuing education purposes.

Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a promising therapeutic approach for several cancers. Despite the growing interest, the widespread use of CAR T-cell therapies has been restricted due to their high cost and the challenges that arise when compared with traditional methods for manufacturing and product QC. Developing sensitive and reliable analytics to characterize CAR-T products' critical quality attributes (CQAs) and tailoring the cell expansion process to achieve these CQAs provides tools for a more consistent manufacturing process.

Join Dr. Laurissa Ouaguia, Scientist, Reagents and Application Development, Agilent Technologies, as she discusses Agilent’s portfolio of cell analysis tools. She will also demonstrate how they can be applied during the production of CAR T-cells using the functionally closed and automated Lonza Cocoon® Platform for cell manufacturing. This approach provides a deeper understanding of the immune phenotype, metabolic fitness, and cytotoxic function of the CAR T-cells throughout the production, allowing for more informed process development.

Key learning objectives

• Explore the Agilent Seahorse XF Analyzer for real-time live-cell metabolic assay and the impedance-based xCELLigence RTCA eSight system for real-time immune cell killing potency assay.
• Understand how Agilent cell analysis platforms deliver reliable, reproducible, and sensitive analytics, providing a deeper understanding of CAR T-cells' immune phenotype, metabolic fitness, and cytotoxic function throughout the production in an automated cell manufacturing platform
• Discover how enhanced analytics can provide valuable tools for in-process analysis during CAR T-cell manufacturing, potentially shortening production timelines and reducing associated costs

Who should attend?

• Researchers and scientists specializing in cell therapy
• Experts in cell therapy analytics and process development
• Cell therapy manufacturing specialists
• Quality control (QC) professionals in the cell therapy field
• Professionals in the pharmaceutical and biopharmaceutical industries focusing on cell therapy

Certificate of attendance
All webinar participants can request a certificate of attendance, including a learning outcomes summary, for continuing education purposes.

In this video from Agilent Technologies, learn more about how accessibility to appropriate tools enables the progression of science by being able to answer questions in a relevant way. Discover the Agilent Seahorse XF Pro Analyzer which has enabled the research of metabolism during cell equilibrium, transforming the ability to address discrete metabolic phenotypes and correlate them with cancer malignancy.

Explore innovations such as oligonucleotide therapeutics, bi-specific antibodies, CAR-T therapy and CRISPR

Advancing drug discovery and development through cutting-edge innovations

Discover how bispecific-antibody-armed, metabolically enhanced headless CAR T-cells mediate high levels of cytotoxicity directed at solid tumors