Cell Metabolism Assays. for OMICS. Research

Transcription

1 Gene-Protein-metabolism links Cell Metabolism Assays for OMICS Research

2 STANDARD Parameters of Functional METABOLIsm Genomics Cells use gene expression to synthesize proteins and other products that are essential for various cellular functions. Gene expression levels, while associated with metabolism, cannot solely predict the functional impact on metabolism. This creates a gap in understanding the complex physiological relation between genes and metabolism. Researchers are using XF Technology to link functional metabolism to gene expression for a complete picture. The XF Cell Mito Stress Test measures the key parameters of mitochondrial respiration: basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. The XF Glycolysis Stress Test measures the key parameters of glycolytic activity: glycolysis, glycolytic capacity, glycolytic reserve. (pmol/min) Control Hdac9 deleted Basal Oligomycin FCCP Mouse T cells XF Assay reveals a link between histone deacetylase 9 (Hdac9) deletion and mitochondrial respiration correlating to a genetic and functional increase in T regulatory cells. (Beier UH et al.,. FASEB J.) (pmol/min/mg protein) MELAS-iPS Basal Respiration MELAS-iPS P<. MELAS-iPS MELAS-iPS ATP Production MELAS-iPS P<. MELAS-iPS MELAS-iPS Maximal Respiration MELAS-iPS P<. MELAS-iPS Spare P<. MELAS-iPS MELAS-iPS MELAS-iPS MELAS-iPS MELAS-iPS MELAS-iPS Rotenone & Oligomycin FCCP antimycin A 7 Human neuronal progenitor cells Relative Expression Levels... MELAS-IPS MELAS-IPS MELAS-IPS OCT SOX PAX NANOG NESTIN XF Cell Mito Stress Test predicts differentiation status in neuronal progenitor cells (NPCs) derived from patients with mutations causing mitochondrial encepharopathy and stroke-like episodes (MELAS) which correlates to gene expression. (Ma H et al.,. Nature.)

3 THE WORLD S MOST ADVANCED METABOLIC ANALYZERS Proven Technology for Cutting Edge Research There are over, references utilizing XF Technology published in leading journals such as Nature and Cell. Scientists are embracing XF Technology to identify metabolic phenotypes and reprogramming to target metabolic pathways for therapeutic purposes. Pyruvate Lactate IV III II GLYCOLYSIS ECAR (Extracellular Acidification Rate) MITOCHONDRIAL RESPIRATION OCR (Oxygen Consumption Rate) I Cells generate ATP Mitochondria consume via glycolysis independent oxygen when oxidizing fatty of oxygen, producing lactic acid acids or other substrates and protons. XF Analyzers measure to generate ATP. XF glycolysis by measuring the extracel- Analyzers measure mitochondrial lular acidification rate (ECAR) of respiration by measuring the oxygen cells. consumption rate (OCR) of cells. Proteomics Advances in proteomic technology enables genome-wide data analysis, pinpointing specific proteins and expression levels. Proteomic data directly links to metabolism without requiring the regulatory mechanisms associated with mrna expression and final protein expression. However, conclusions utilizing only these types of experiments are limited. XF Technology not only confirms proteomic data but also allows for further investigation of protein function. WT PKA-deficient ATP-linked Respiration Rate (pmol O /min/ x cells) CPT-cAMP Murine lymphoma cells G Oligomycin -DG Oligomycin -DG.7-.9 Not represented G G G Microarray Fold Change Oligomycin GO_BP: Gene & Protein Expression Profile metabolic process mrna mrna -DG RPIA PYGL PFKP LDHB GYS FABP ENO ALDOC 9 G 8 Time (min)oligomycin -DG 9 8 Time (min) protein protein relative expression level G/ Murine bone-derived macrophages XF Cell Mito Stress Test reveals that protein kinase A (PKA)dependent changes in protein and gene expression correlate with changes in ATP-linked respiration. XF Glycolysis Stress Test reveals the increased glycolytic capacity of granulocyte-stimulated macrophages (), confirming the inherently higher glycolytic protein expression. (Wilderman A et al.,. J Cell Biology.) (Na YR et al.,. Mol Cell Proteomics.)

4 7,, 7,,,, 7,, 8 9, MEASURING THE KEY PARAMETERS OF Functional Cell metabolism Metabolomics Techniques, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), enable the analysis of hundreds of metabolites in a given sample. However, the data from these techniques alone are not sufficient to assess functionality within a biological context. XF Technology provides real-time analysis of cellular response to stress or nutrients, and can direct metabolomics research, adding efficiency and direction to any area of research. XF Glycolysis Stress Test predicts sensitivity to first-line EGFR inhibitors used in lung adenocarcinoma cells, and correlates to reduced metabolite levels. (Makinoshima H et al.,. J Biol Chem.) Extracellular Extracelluar Acidification Rate Rate (ECAR) (ECAR) Oxygen Oxygen Consumption Consumption Rate (OCR) Rate (OCR) (pmol/min) (pmol/min) (pmol/min) GEF= Gefitinib ERLO= Erolotinib DMSO GEF ERLO DMSO GEF ERLO HCC87 PC9 H97 Human lung adenocarcinoma cells Th Th7 Treg Oligomycin Oligomycin -DG -DG 7 7 Rotenone & Oligomycin FCCP antimycin Rotenone A & Oligomycin FCCP antimycin A 8 8 Glycolysis Glycerol P Lactate GP/FP Fructose,-P DHAP,-P Glycerate Glycerate P PEP Pyruvate 8 P Gluconate Oxidative PPP Th Th7 Treg Relative level normalized to naive (red line) Nonoxidative PPP GP Eryhrose Sedoheptulose P 7P... Cis- α-keto.. Aconitate glutarate... TCA cycle Citrate Succinate.. Oxaloacetate Fumarate Acetyl CoA Malate 7 Time Murine T cells 7 XF Glycolysis Stress Test and XF Cell Mito Stress Test reveal increased levels of mitochondrial respiration and glycolytic activity in TH7 (T cells) which correlate to changes in metabolite levels. (Gerriets VA et al.,. J Clin Invest.) DMSO GEF ERLO pmol/ cells pmol/ cells pmol/ cells pmol/ cells FP DHAP ATP GP FBP pmol/ cells GLUTs HKs GP EP pmol/ cells pmol/ cells PG XP RuP RP PG pmol/ cells pmol/ cells pmol/ cells pmol/ cells PEP PKMs LDHs Lactate Pyruvate PDHs pmol/ cells Acetyl-CoA What OUR Customers ARE SAYING We were really surprised by the impact of measuring metabolism in our model system. This opens up new research avenues for us. - Researcher at Rosalind Franklin Medical School With very little learning curve, XF Technology provides highquality data. We generated publishable results within the first month. - Researcher at Case Western Reserve University When we decided to more closely investigate branched - chain amino acid metabolism, we chose to incorporate our XF Technology, knowing that it is a powerful tool for assessing mitochondrial function and metabolism. - Researcher at University of California, San Diego

5 Functional metabolic assays The Genotype-Phenotype Link The traditional paradigm encompassing the flow of information from gene to protein to function can be found in nearly every biology and biochemistry textbook. At the time, this discovery was thought to explain all of biology, health, and disease. However, only focusing on the expression of a single gene or protein neglects the multi-faceted consequences of that expression on metabolism, as well as overall cell and tissue health. Mitochondria are at the crux of cellular energy generation, intracellular signaling, and cell death and survival regulation. Compromised mitochondria can strain the expansive and intricate cellular metabolic network. There is increasing appreciation for the role of mitochondria in disease pathology, etiology, and cell health. Gene Expression Integrating processes that demonstrate the link amongst genes, proteins, and metabolism allows researchers to analytically answer their scientific questions. XF Technology provides researchers the necessary tools to measure the functional, metabolic impact of gene and protein expression, faster and easier using live cells. Functional Metabolism Full Picture of Cell Health Protein Expression GENES & METABOLISM LINK GLUCOSE substrates and metabolism GLUCOSE FATTY ACID PI/AKT HIF GLYCOLYSIS p MYC LACTATE PYRUVATE FATTY ACID CITRATE MYC HIF Acetyl-CoA HIF PYRUVATE LIPID SYNTHESIS TCA TCA PI/AKT ETC GLUTAMATE MYC GLUTAMINOLYSIS GLYCOLYSIS (ECAR) MITOCHONDRIAL RESPIRATION (OCR) GLUTAMINE

6 Gold Standard metabolic Assays MEASURING THE KEY PARAMETERS OF CELL METABOLISM (pmol/min) XF Cell Mito Stress Test Profile 8 8 Mitochondrial Respiration Oligomycin Basal Respiration ATP Production FCCP Rotenone & antimycin A Maximal Respiration Proton Leak Non-mitochondrial Oxygen Consumption Time (minutes) XF Cell Energy Phenotype Test Metabolic Phenotype & Potential Mitochondrial Respiration Aerobic Baseline Phenotype Quiescent Metabolic Potential Glycolysis Energetic Stressed Phenotype Glycolytic Spare % GLC+GLN+FA Fuel Oxidation 8 XF Glycolysis Stress Test Profile Glycolytic Function Oligomycin -DG Glycolytic Reserve Glycolytic Glycolysis Non-glycolytic Acidification Time (minutes) XF Mito Fuel Flex Test Profile Mitochondrial Function Dependency Flexibility Dependency Pathway Glutamine Pathway Fatty Acid Pathway Pathway Glutamine Pathway Fatty Acid Pathway Flexibility Dependency Corporate Headquarters European Headquarters Asia-Pacific Headquarters Seahorse Bioscience Inc. Esquire Road North Billerica, MA 8 US Phone: Seahorse Bioscience Europe Fruebjergvej Copenhagen DK Phone: Seahorse Bioscience Asia 99 Guo Shou Jing Rd, Suite 7 Pudong, Shanghai CN Phone: Rev All rights reserved. Seahorse Bioscience and the Seahorse logo are trademarks of Seahorse Bioscience Inc. The content in this brochure is for informational purposes only and may be subject to change without notice.