Agilent Technologies: Seahorse XF Introduction

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1 Agilent Technologies: Seahorse XF Introduction Tools for investigating cell metabolism diagnostic procedures. 1

Glucose Oxidation of: Glucose Fat Amino Acids Metabolism is

2 The Engines Cells Generate Energy via Two Metabolic Pathways Glycolysis [ H + ] Respiration [ Oxygen ] Fermentation of Glucose Oxidation of: Glucose Fat Amino Acids Metabolism is measured via the rates of change in ph and oxygen concentration

3 Measuring functional metabolism with the Seahorse XF Analyzer Oxygen Consumption Rate (OCR): Measurement of mitochondrial respiration Extracellular Acidification Rate (ECAR): Indicator of glycolysis

4 Measuring Mitochondrial Respiration and Glycolysis in a Microplate Sensor Probes Fiber Optics XF Cell Culture Microplate Assay Medium Solid State Sensor Cell Monolayer Well 1 Well 2

Measuring Mitochondrial Respiration and Glycolysis in a Microplate The sensor bio- cartridge is A transient raised, bringing microchamber is the the system system

5 Measuring Mitochondrial Respiration and Glycolysis in a Microplate The sensor bio- cartridge is A transient raised, bringing microchamber is the the system system back to back baseline to formed baseline The transient micro-chamber enables rapid measurement of mitochondrial respiration and glycolysis in minutes Well 1 Well 2

6 Measuring mitochondrial respiration and glycolysis in a microplate with live cells MIX Gentle up/down 3 minutes standard Restores O2 and ph levels WAIT Probes remain Up Only in 24-well protocols Cycle ~ 3 times MEASURE Probes down- microchamber formed 2-3 minutes standard INJECT Manifold system injects all ports of a given position A or B etc. then Cycle

7 Sequence of Operations and Raw Data: ph vs. time ECAR MEASURE CYCLE INJECT

8 Automatic Calculation of Extracellular Acidification Rate (ECAR)

9 Sequence of Operations and Raw Data: O 2 vs. time OCR

10 Automatic Calculation of Oxygen Consumption Rate (OCR)

11 Measuring functional metabolism with the Seahorse XF Analyzer Every Seahorse XF Analyzer performs automated compound injections enabling the stimulation of cellular metabolism in real time

12 Automated compound injections enable kinetic, functional data 4 compound injection ports per well Add inhibitors, stimulants, substrates, etc. Injections are defined by the user

13 Seahorse XF Analyzers and Standardized Seahorse XF Assays XFp Analyzer 8 well XF Glycolytic Rate Assay XFe24 Analyzer 24 well XF Cell Energy Phenotype Test XF Mito Fuel Flex Test XFe96 Analyzer 96 well

14 Measuring functional metabolism with the Seahorse XF Analyzer Combining simultaneous measurement of OCR and ECAR with automated compound injections allows for powerful analysis of cell metabolism

15 Oxygen Consumption Rate (OCR) Mitochondrial Respiration Metabolic Engines: XF Energy Map XF Energy Map Aerobic Energetic Define XF Metabolic Phenotypes Passage 12 Activated T cells Monitor Cell Quality and Health Neurons Quiescent Memory T Cells Passage 36 Lymphoma Cells Glycolytic Glycolysis Extracellular Acidification Rate (ECAR)

16 Oxygen Consumption Rate (OCR) Mitochondrial Respiration Metabolic Engines: XF Energy Map XF Energy Map Aerobic Energetic Define XF Metabolic Phenotypes Activated T cells Monitor Cell Quality and Health Neurons Kidney Cells See XF Metabolic Switches Fh1-/-CL1 Kidney Cells Lymphoma Cells Memory T Cells Quiescent Glycolytic Glycolysis Extracellular Acidification Rate (ECAR)

Activated T cells Monitor Cell Quality and Health See XF Metabolic Switches Neurons Measure XF Metabolic

17 Oxygen Consumption Rate (OCR) Mitochondrial Respiration Metabolic Engines: XF Energy Map and XF Cell Energy Phenotype Test XF Energy Map Aerobic Stressed Neurons Energetic Define XF Metabolic Phenotypes Activated T cells Monitor Cell Quality and Health See XF Metabolic Switches Neurons Measure XF Metabolic Potential Lymphoma Cells Memory T Cells Quiescent Glycolytic Glycolysis Extracellular Acidification Rate (ECAR)

Glycolysis for ATP production FCCP: Uncouple oxygen consumption

18 Seahorse XF Cell Energy Phenotype Test Activate both pathways: Oligomycin: Block mitochondrial ATP synthesis, force cells to use Glycolysis for ATP production FCCP: Uncouple oxygen consumption from ATP synthesis, push complex IV to attempt to restore membrane potential

19 OCR (pmol/min) OCR (pmol/min) Seahorse XF Cell Energy Phenotype Test In-vitro tumor phenotyping Ex-vivo tumor cells Aerobic Energetic Aerobic Energetic Quiesce Glycolytic ECAR (mph/min) 0 Quiesce Glycolyti ECAR (mph/min) Normal Astrocyte Glioblastoma (T98G) Glioblastoma (U251) Low grade tumor High grade tumor

20 Measuring functional metabolism with the Agilent Seahorse XF Analyzer

21 Agilent Seahorse XF Cell Mito Stress Test for Mitochondrial Function Patient Fibroblasts Parkinson s Disease Lower spare capacity in patient Data courtesy of James Bennet Lab, University of Virginia

22 Agilent Seahorse XF Cell Mito Stress Test for Mitochondrial Function

OCR (% baseline) CARDIOVASCULAR Stress Test Sensitivity Reveals Sub-Lethal Stress Response and Mechanism 300 250 200 0 µm HNE 5 µm HNE 10 µm HNE 20 µm HNE XF Cell Mito Stress Test Oligo FCCP

23 OCR (% baseline) CARDIOVASCULAR Stress Test Sensitivity Reveals Sub-Lethal Stress Response and Mechanism µm HNE 5 µm HNE 10 µm HNE 20 µm HNE XF Cell Mito Stress Test Oligo FCCP Antimycin A 150 HNE Time (min) Neonatal Rat Ventricular Myocytes Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal Bradford G. Hill, Brian P. Dranka, Luyun Zou, John C. Chatham and Victor M. Darley-Usmar, Biochem J Nov 15;425(1):99-107

, (2016) Cancer Res Seahorse XF Glycolysis Stress Test reveals a

24 Agilent Seahorse XF Glycolysis Stress Test Glycolytic Response to Energy Demand SKBR3 cells +/- immuno-regulatory protein Lim, S et al., (2016) Cancer Res Seahorse XF Glycolysis Stress Test reveals a switch to glycolysis driven by over expression of immuno-regulatory protein B7-H3.

CANCER XF Glycolysis Stress Test Measures Glycolytic Phenotype Susceptible to Buffer Therapy PC-3M PCS Prostate Tumor Cells Primary Prostate

25 CANCER XF Glycolysis Stress Test Measures Glycolytic Phenotype Susceptible to Buffer Therapy PC-3M PCS Prostate Tumor Cells Primary Prostate Epithelial Cells Free Base Lysine Increases Survival and Reduces Metastasis in Prostate Cancer Model Ibrahim-Hashim, et al., J Cancer Sci Ther 2011, S1

26 Agilent Seahorse XF Glycolytic Rate Assay for Glycolysis

27 Account for CO 2 -dependent acidification GLYCOLYSIS MITOCHONDRIAL RESPIRATION Glucose 2-DG Rotenone H + Antimycin A H + H + H + hexokinase Glucose-6-Phosphate I II III IV V ADP ATP Pyruvate MITOCHONDRIA NADH H + H + O 2 H + ADP H 2 O H + ATP Lactate + H + CO 2 + H 2 O HCO 3- + H + TCA NADH ATP CO 2

28 Glycolytic Rate Assay : Determining the Rate Convert ECAR to PER (using BF and Kvol) Calculate mitoper (using CCF and OCR) Subtract mitoper from PER to obtain glycoper MCF-7 ctrl MCF-7 DOX

29 A431 cells: Highly glycolytic AA/Rot 2-DG 92% glycolytic acidification mito OCR Report Generator A431 cells (40K/well) ECAR (mph/min) 50 ±1 PER (pmol/min) 475 ±4 glycoper (pmol/min) 437 ± 4

30 Bovine A431 Aortic cells: Highly Endothelial glycolytic cells (BAEC) : Highly oxidative AA/Rot 2-DG 53% glycolytic acidification Report Generator BAEC (50K/well) ECAR (mph/min) 24 ±1 PER (pmol/min) 220 ±9 glycoper (pmol/min) 117 ± 6

31 XF Glycolytic Rates Directly Correlate with Lactate Accumulation Basal lactate measured as total lactate (nmol) accumulated in extracellular media during 1 hour (lactate kit) and converted to glycolytic proton efflux rate, glycoper (pmol/min) Slope = 1.0 R 2 = 0.90 n = 53 (10 cell lines) Basal glycoper = Basal Lactate

32 induced XF Glycolytic Rate Assay: Two Assay Schemes basal compensatory basal compensatory Basic Assay Induced Assay GlycoPER (pmol/min) Rot/AA 2-DG GlycoPER (pmol/min) Acute injection Rot/AA 2-DG Time (minutes) Time (minutes)

33 XF Glycolytic Rate Assay Report Generator outputs

34 XF Glycolytic Rate Assay Report Generator outputs MCF7 Ctrl MCF7Dox Parameter Units Average Average Basal Glycolysis pmol/min Basal Proton Efflux Rate pmol/min % PER from glycolysis % 59% 70% Compensatory Glycolysis pmol/min mitoocr/glycoper Post 2-DG acidification pmol/min 81 99

35 Agilent Seahorse XF Mito Fuel Flex Test for Mitochondrial Fuel Oxidation Seahorse XF Mito Fuel Flex Test

36 OCR (pmol/min) Measuring the 3 pathways that fuel mitochondria Glucose Fatty Acids Glutamine nucleus mitochondria For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

37 OCR (pmol/min) 100% GLC + GLN + FA Oxidation Measuring the 3 pathways that fuel mitochondria Glucose Fatty Acids Glutamine BPTES UK5099 Etomoxir nucleus mitochondria For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

38 % GLC + GLN + FA Ox Measuring the 3 pathways that fuel mitochondria Glucose Fatty Acids Glutamine nucleus mitochondria For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

39 % GLC + GLN + FA Ox Are my cells dependent on the glucose pathway? Glucose Fatty Acids Glutamine UK5099 nucleus mitochondria For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

40 % GLC + GLN + FA Ox Are my cells dependent on the glucose pathway? Yes! Glucose Fatty Acids Glutamine UK5099 nucleus 100 Glucose Dependent 50 0 mitochondria For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

41 Fuel Oxidation (% GLC + GLN + FA Oxidation) Fuel Dependency Fuel Dependency The measurement of cells reliance on a particular fuel pathway to maintain baseline respiration For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

42 % GLC + GLN + FA Ox What is the capacity of the Glucose pathway? Glucose Fatty Acids Glutamine Etomoxir BPTES nucleus Glucose Capacity 0 mitochondria For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

43 Fuel Oxidation (% GLC + GLN + FA Oxidation) Fuel Capacity Fuel Dependency The measurement of cells reliance on a particular fuel pathway to maintain baseline respiration. Fuel Capacity The cells ability to use a fuel pathway to meet metabolic demand when other fuel pathways are inhibited For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

44 Fuel Oxidation (% GLC + GLN + FA Oxidation) Fuel Flexibility Flexibility Dependency Fuel Dependency The measurement of cells reliance on a particular fuel pathway to maintain baseline respiration. Fuel Capacity The cells ability to use a fuel pathway to meet metabolic demand when other fuel pathways are inhibited. Fuel Flexibility The ability of cells to increase oxidation of a particular fuel in order to compensate for inhibition of alternative fuel pathways. The difference is flexibility For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

Seahorse XF Mito Fuel Flex Test For researchers seeking to understand which substrates their cell models are using for energy production, the Seahorse XF Mito Fuel Flex Test determines

45 Seahorse XF Mito Fuel Flex Test For researchers seeking to understand which substrates their cell models are using for energy production, the Seahorse XF Mito Fuel Flex Test determines both dependency and flexibility of cells to oxidize glucose, glutamine, and/or fatty acids in less than two hours, without the need for nutrient starvation. Seahorse XF Mito Fuel Flex Test

Kits available for all Seahorse XF Analyzers Sufficient reagents for 6 assays

Pathway Specific Target UK5099 Glucose Blocks the mitochondrial pyruvate

Etomoxir (ETO) Fatty Acid Blocks carnitine palmitoyltransferase 1A (CPT1A) For

46 Kits available for all Seahorse XF Analyzers Sufficient reagents for 6 assays per kit (Etomoxir) (UK5099) (BPTES) Inhibitors and mechanism of action Fuel Pathway Specific Target UK5099 Glucose Blocks the mitochondrial pyruvate carrier (MPC) BPTES Glutamine Allosteric inhibitor of glutaminase (GLS1) Etomoxir (ETO) Fatty Acid Blocks carnitine palmitoyltransferase 1A (CPT1A) For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

47 Example: Assay for glutamine dependency BPTES Eto/UK % of GLC, FA, and GLN oxidation Baseline OCR BPTES inhibited OCR BPTES + Eto + UK5099 inhibited OCR For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

48 Calculating Dependency (Glutamine example) BPTES UK5099/ETO [ %Dependency = Rate 3 Rate 9 Rate 3 Rate 15 [ X 100 Inhibitors and mechanism of action Fuel Pathway Specific Target UK5099 Glucose Blocks the mitochondrial pyruvate carrier (MPC) BPTES Glutamine Allosteric inhibitor of glutaminase (GLS1) Etomoxir (ETO) Fatty Acid Blocks carnitine palmitoyltransferase 1A (CPT1A) For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

49 Calculating Capacity (Glutamine example) UK5099/ETO BPTES [ %Capacity = 1 - Rate 3 Rate 9 Rate 3 Rate 15 [ X 100 Inhibitors and mechanism of action Fuel Pathway Specific Target UK5099 Glucose Blocks the mitochondrial pyruvate carrier (MPC) BPTES Glutamine Allosteric inhibitor of glutaminase (GLS1) Etomoxir (ETO) Fatty Acid Blocks carnitine palmitoyltransferase 1A (CPT1A) For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

50 Calculating Flexibility (Glutamine example) %Flexibility = %Capacity - %Dependency For For Research Research Purposes Use Only. Not Not for for diagnostic use in purposes. 3/28/2018

51 % Dependency Seahorse XF Mito Fuel Flex Test for Mitochondrial Fuel Oxidation Seahorse XF Mito Fuel Flex Test A549 Fuel Dependency normoxia v. hypoxia GLC-Normoxia GLC-Hypoxia GLN-Normoxia GLN-Hypoxia LCFA-Normoxia LCFA-Hypoxia

52 XF Palmitate-BSA FAO Substrate Reliable, functional fatty acid oxidation assays are easy with a ready-to-use, bioavailable Palmitate-BSA conjugate

53 OCR (pmol/min) Analysis of FAO used as a substrate in a Cell Mito Stress Test XF FAO Assay Parameters Basal Respiration FAO of Exogenous Palmitate FAO of Endogenous Fatty Acids Maximal Respiration Oligo FCCP Rtn/AA FAO of Exogenous Palmitate FAO of Endogenous Fatty Acids time (minutes) Control Control, +Eto Palmitate Palmitate, +Eto

Seahorse XF Fatty Acid Oxidation Assay reveals

metabolism to enable long-term survival by

54 Seahorse XF Fatty Acid Oxidation Assay reveals T-cell metabolic reprogramming following infection Following viral infection of the skin, resident T-cells reprogram their metabolism to enable long-term survival by increasing fatty-acid uptake and oxidative metabolism. Pan 2017 Nature

XF Plasma Membrane Permeabilizer (PMP) XF PMP

analysis in small, clinically relevant samples

Thiazolidinediones are acute, specific inhibitors

55 XF Plasma Membrane Permeabilizer (PMP) XF PMP permeabilizes the plasma membrane without damaging the mitochondria. C2C12 Myoblast MCP1 & MCP2 Knockdowns Mechanistic analysis in small, clinically relevant samples Mechanistic analysis in genetically modified cells Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier Divakaruni AS and Murphy AN, PNAS (2013) 110:

56 Metabolic change is an early step in immune cell response T cells ramp glycolysis instantly following activation Pearce 2013 Science

XF T-cell activation assay reveals early, kinetic window In-Seahorse Activation 6 minutes CD69 Flow Cytometry 5 hours IFN-γ ELISA 12 hours (min) CD8+ T

57 XF T-cell activation assay reveals early, kinetic window In-Seahorse Activation 6 minutes CD69 Flow Cytometry 5 hours IFN-γ ELISA 12 hours (min) CD8+ T cells Gubser, PM et al Rapid effector function of memory CD8 T cells requires an immediate-early glycolytic switch. Nature Immunology 14(10):

58 T cell activation as measured by Seahorse reflects the biological outcome Naive Memory Naive Memory Gubser 2013 Nat Immunol

59 The consequence of decreased activation Decreased activation predicts lower proliferation rate Proliferation measured after 3 days Gubser 2013 Nat Immunol

60 Real time metabolic analysis delivers a rapid and robust indicator of activation in minutes Use injection ports to investigate modulators of T cell activation kinetics

61 A new approach to quantifying macrophage activation PBMC-derived macrophages

62 Macrophage activation quantified by XF analysis predicts cytokine production

63 RAW macrophages have biphasic activation Mechanism of action discovery LPS TLR4 1400W inos Nitric Oxide

64 Review from EMBO Figure 1. Influence of energy metabolism on pluripotent status diagnostic procedures.

70 Reprogramming efficiency is improved with increased glycolysis and oxidative phosphorylation Sone 2017 Cell Metabolism

Reprogramming efficiency is improved with increased glycolysis and oxidative phosphorylation Day 6, 47 genes expression levels were higher with ipsc committed cells, distilled to 16 DNA binding TFs

71 Reprogramming efficiency is improved with increased glycolysis and oxidative phosphorylation Day 6, 47 genes expression levels were higher with ipsc committed cells, distilled to 16 DNA binding TFs Zic3 + Esrrb = highest reprogramming efficiency Overexpression of Zic3 activates glycolytic metabolic pathway Esrrb reinforces this pathway in reprogramming Esrrb restores cellular respiration ability and enhances reprogramming Sone 2017 Cell Metabolism

Quantifying Cellular ATP Production Rate Using Agilent Seahorse XF Technology

White Paper Quantifying Cellular ATP Production Rate Using Agilent Seahorse XF Technology Authors Natalia Romero George Rogers Andy Neilson Brian P. Dranka Agilent Technologies, Inc., Lexington, MA, USA