Cell Metabolism Assays. for. Immunology. Research

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1 the immunity-metabolism link Cell Metabolism Assays for Immunology Research

2 GOLD STANDARD ASSAYS FOR MEASURING METABOLIC RePROGRAMMING Metabolic Signatures Cell activation, amplification, and effector function are crucial aspects of the immune cell life cycle. Researchers are using XF technology to examine the cross-talk between immune cells and their metabolic signatures to gain insight into disease pathology, etiology, and possible treatment options. The XF Cell Mito Stress Test measures the key parameters of mitochondrial function: basal respiration, ATP-linked respiration, proton leak, maximal respiration, and spare respiratory capacity. The XF Glycolysis Stress Test measures the three key parameters of glycolytic function: glycolysis, glycolytic capacity, and glycolytic reserve FCCP Rotenone/ M M1 M2 (% mph/min) Glucose ** ** Uninfected HIV-infected (pmol/min/μg protein) Platelets Monocytes Lymphocytes Basal Neutrophils (mph/min/μg protein) Murine bone-derived macrophages Human s Human platelets and leukocytes Huang SC et al., (214) Nat Immunol. Hegedus A et al., (214) Retrovirology Kramer PA et al., (214) Redox Biol. XF Cell Mito Stress Test profiles metabolic signatures of activated bone marrow-derived macrophage subtypes. XF Assay reveals the role of glycolysis in HIV infection. XF Metabolic Switch Assay reveals distinct metabolic signatures in platelets and leukocytes. Metabolic reprogramming Signaling plays an important role in eliciting immunological response by coordinating immune cell communication and actions. Researchers are using XF technology to probe the signaling and metabolic programming of immune cells FCCP WT PBS WT Poly IC Rotenone/ ECAR IFNAR-/- Poly IC IFNAR-/- Poly PBS WT IFNAR -/ Inhibitor or DMSO α-cd3 and α-cd DMSO Events (% max) Activated Activated + PI(3)K inhibitor efluor 67 PI(3)K/Akt inhibitor ECAR Rapa 4 2 Unstim - - IL2/12 * + Murine dendritic cells Human s Murine natural killer cells Pantel A et al., (214) PLoS Biol. Gubser PM et al., (213) Nat Immunol. Donnelly RP et al., (214) J Immunol. XF Cell Mito Stress Test identifies IFNAR requirement for metabolic reprogramming. Real-time activation of memory s demonstrates a connection amongst signaling pathways, glycolysis, and proliferation. XF Assay reveals Natural Killer (NK) cell dependence on mtorc1 following stimulation of IL-2 and IL-12.

THE WORLD S MOST ADVANCED METABOLIC ANALYZERS Proven Technology for Cutting Edge Research There are over

Scientists are embracing XF technology to identify metabolic phenotypes and reprogramming to target

Glucose Pyruvate Lactate IV III II I Mitochondrial Respiration Glycolysis ECAR (Extracellular

the immune system is to protect the host by targeting foreign antigens, controlling or clearing

However, metabolic interventions present a largely untapped area of disease research.

antigen Baseline Induced 2 2 6 αcd3/cd28 1 Control 6 ** 4 2 Th17 TGFβ1 Donor cells (per ml of blood)

Rotenone/ Control 4 1 Days 1 2 2, 1, *** Th17 2x1 1x1 3º Day 7 Recall FCCP 1º Day 7 Recall Rotenone 1 1,

IL1β 7 3º 1º Naive 2 4 6 1º Day 7 8 1 Human s Murine T helper (Th) cells Murine s Yin Y et al.

3 THE WORLD S MOST ADVANCED METABOLIC ANALYZERS Proven Technology for Cutting Edge Research There are over 2, 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. Glucose Pyruvate Lactate IV III II I Mitochondrial Respiration Glycolysis ECAR (Extracellular Acidification Rate) OCR (Oxygen Consumption Rate) Metabolic Effect of Therapeutics The primary role of the immune system is to protect the host by targeting foreign antigens, controlling or clearing infections, and attacking cancerous cells. However, metabolic interventions present a largely untapped area of disease research. XF technology provides the necessary tools to understand the effects of therapeutic candidates on the antigen Baseline Induced αcd3/cd28 1 Control 6 ** 4 2 Th17 TGFβ1 Donor cells (per ml of blood) Th17IL1β 2 1 pmol O2/min 4 Exrtacellular Acicification Rate (ECAR) 6 8 Th17 TGFβ1 3 αcd3/cd28 FCCP Rotenone/ Control 4 1 Days 1 2 2, 1, *** Th17 2x1 1x1 3º Day 7 Recall FCCP 1º Day 7 Recall Rotenone 1 1, * 4x1 3x1 Tumor size (mm2) of interest, and on the immune system. IL1β 7 3º 1º Naive º Day Human s Murine T helper (Th) cells Murine s Yin Y et al., (21) Sci Transl Med. Chatterjee S et al., (214) Cancer Res. Fraser KA et al., (213) Immunity Real-time activation reveals metabolic signatures of lupus patient-derived CD4+ s. XF Assays reveal a metabolic switch which correlates to the antitumor ability of Th17IL1β cells. 12 3º Day 7 XF Assays reveal that tertiary immunization increases memory CD8+ recall.

MEASURING THE KEY PARAMETERS OF Immunometabolism Substrate Utilization, Flexibility & Dependency Each immune cell type has a specific function within the immune system.

4 MEASURING THE KEY PARAMETERS OF Immunometabolism Substrate Utilization, Flexibility & Dependency Each immune cell type has a specific function within the immune system. Cell fate decisions, activation, amplification, and effector function are driven by metabolism. To maintain the energy demands of the cell at each stage of its life cycle, the metabolic requirements for substrates or fuels that feed into the metabolic engines are altered. XF technology provides the capability to examine the effect of substrate utilization and dependency, yielding powerful metabolic data Glucose 2-DG Peg-BSA Peg-Arg I % s proliferating (CFSE) ***. 1 (μg/ml pegylated protein) 2 1 **** glucose ns + glucose Control Rapamycin Murine s Fletcher M et al., (21) Cancer Res. Murine s Goldberg EL, et al., (214) J Immol. XF Glycolysis Stress Test identifies L-Arginine dependence for proliferation in s. XF Assay reveals glucose-insensitivity of naïve s in the presence of rapamycin control or etomoxir PMA/ionomycin 1 1 B-ALL cell line #1 B-ALL cell line #2 B-ALL cell line #3 B cell line #1 B cell line #2 B cell line #3 No Fuel Glutamine Glutamine + Glucose Murine s Human B cells van der Windt GJ et al., (213) PNAS Liu T et al., (214) Cell Death Dis. XF Assay reveals a role of fatty acid oxidation in memory response. XF Assay reveals a glucose dependence in B-cell acute lymphoblastic leukemia (B-ALL) cells.

Functional XF metabolic assays - THE intersection of immunology & metabolism XF Technology provides the tools TO MEASURE IMMUNOMETABOLISM Immunologists study how the innate and adaptive immune system

5 Functional XF metabolic assays - THE intersection of immunology & metabolism XF Technology provides the tools TO MEASURE IMMUNOMETABOLISM Immunologists study how the innate and adaptive immune system recognizes and responds to insults. Whether the research focus is on a specific immune cell type, signaling pathway, or disease area, immunologists are actively exploring the mechanisms that drive and perpetuate antigen recognition and response. Immune system metabolism (referred to as immunometabolism ) has emerged as a key mechanism in understanding the connection between metabolic pathways and immune responses. Each immune cell type has a specialized role in an immune response, as well as a preferred metabolic pathway to generate energy required to maintain homeostasis. Research into metabolic changes provides insight into metabolic signature, signaling, and substrate preference. These metabolic choices are leading to new opportunities to modulate immunological response for therapeutic intervention. Signaling Pathways Cell Type Metabolism Disease Area XF technology is at the forefront, providing powerful and effective tools to explore the burgeoning field of immunometabolism. substrates and metabolism GLUCOSE FATTY ACID LACTATE PYRUVATE FATTY ACID CITRATE Cell LINEAGE and immunometabolism Acetyl-CoA TCA GLUTAMATE Naive Activated Fatty acid oxidation Effector Mitochondrial respiration (OCR) Glycolysis (ECAR) Memory GLYCOLYSIS (ECAR) ETC MITOCHONDRIAL RESPIRATION (OCR) GLUTAMINE

6 Gold Standard metabolic Assays MEASURING THE KEY PARAMETERS OF CELL METABOLISM XF Cell Mito Stress Test Profile Mitochondrial Respiration Basal Respiration ATP Production FCCP Rotenone & Maximal Respiration Proton Leak Non-mitochondrial Oxygen Consumption Spare Time (minutes) XF Cell Energy Phenotype Test Metabolic Phenotype & Potential Mitochondrial Respiration Aerobic Baseline Phenotype Quiescent Metabolic Potential Glycolysis Energetic Stressed Phenotype Glycolytic % GLC+GLN+FA Fuel Oxidation XF Glycolysis Stress Test Profile Glycolytic Function Glucose Glycolysis Glycolytic 2-DG Non-glycolytic Acidification Glycolytic Reserve Time (minutes) XF Mito Fuel Flex Test Profile Mitochondrial Function Dependency Flexibility Dependency Glucose Pathway Glutamine Pathway Fatty Acid Pathway Glucose Pathway Glutamine Pathway Fatty Acid Pathway Flexibility Dependency Corporate Headquarters European Headquarters Asia-Pacific Headquarters Seahorse Bioscience Inc. 16 Esquire Road North Billerica, MA 1862 US Phone: Seahorse Bioscience Europe Fruebjergvej 3 21 Copenhagen DK Phone: Seahorse Bioscience Asia 199 Guo Shou Jing Rd, Suite 27 Pudong, Shanghai 2123 CN Phone: Rev 3 21 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.

Cell Metabolism Assays. for OMICS. Research

Cell Metabolism Assays. for OMICS. Research Gene-Protein-metabolism links Cell Metabolism Assays for OMICS Research STANDARD Parameters of Functional METABOLIsm Genomics Cells use gene expression to synthesize proteins and other products that are