Introduction to the XF Cell Mito Stress Test Assay

1. O O O O O O O O O 2 2 2 2 2 2 2 2 2 + H + H + H + H + + H H Introduction to the XF Cell Mito Stress Test Assay

2. Presentation Outline How does the XFeAnalyzer measure mitochondrial function using OCR and ECAR? How are researchers using the XF Cell Mito Stress Test Kit to gain further insight in their studies? How are researchers using the XFe Analyzer to study metabolism? Other XF Reagents

3. XFe Analyzers Measure the Two Major Energy Pathways ExtracellularAcidification Rate (ECAR) =Glycolysis Oxygen Consumption Rate (OCR) = MitochondrialRespiration

4. XF Cell Mito Stress Test Measure the key parameters of mitochondrial function: • Basal Respiration • ATP Production • Proton Leak ATPProduction • Maximal Respiration and Spare Capacity Basal Respiration Proton Leak Non-mitochondrial Respiration XF Cell Mito Stress Test Profile Spare Capacity Maximal Respiration Mitochondria have an energy reserve to handle acute stress demands

5. What’s in the XF Cell Mito Stress Test Kit Assay Manual and Protocol Antimycin A Rotenone Oligomycin Quick StartGuide FCCP SW Application + automated results output DMSO Now it’s easy to measure mitochondrial function in cells!

6. Mito Stress Test Assay Medium Using XF Assay Medium (DMEM with NO sodium bicarbonate) Contains Glutamax & low phenol red (3 mg/L) The user adds substrates, such as: Glucose Pyruvate pH 7.4 at 37oC Using XF Base Medium (DMEM with NO sodium bicarbonate) NO Glutamax & low phenol red (3 mg/L) The user adds substrates, such as: Glucose Pyruvate Glutamine (or Glutamax) pH 7.4 at 37oC

7. XF CELL MITO STRESS TESTComponents of Basal Respiration Basal Respiration: • ATP production • Proton Leak • Non-Mito Respiration Basal Respiration

8. XF CELL MITO STRESS TESTOligomycin-sensitive OCR Oligomycin response: • ATP production • Substrate matters ATPProduction Basal Respiration

9. XF CELL MITO STRESS TESTMaximal Respiration FCCP response: • Maximal respiration • Substrate matters ATPProduction Basal Respiration Maximal Respiration

10. XF CELL MITO STRESS TESTSpare Capacity Spare Capacity: • Ability to respond to ATP demand • Substrate matters • Dependent on other respiratory parameters ATPProduction Basal Respiration Spare Capacity Maximal Respiration

11. XF CELL MITO STRESS TESTProton Leak Proton leak: • ETC integrity ATPProduction Basal Respiration Proton Leak Spare Capacity Maximal Respiration

12. XF CELL MITO STRESS TESTNon-Mitochondrial Respiration Non-mitochondrial Respiration: • Cytosolic oxygen consumption ATPProduction Basal Respiration Proton Leak Non-mitochondrial Respiration Spare Capacity Maximal Respiration

13. Experiencing the XF Cell Mito Stress Test • Optimization • Cell seeding titration • Compound titration Stress Test Data Analysis

14. Translational Medicine XF Stress Test Identifies Mitochondrial Dysfunction in Diseases Cell type: Lymphocytes Cell type: Fibroblasts Patient Patient • Parkinson’s Disease • Lower spare capacity in patient • Autism * • Higher spare capacity in patient Data courtesy of James Bennet LabUniversity of Virginia Data courtesy of Sher Hendrickson NCI/NIH Frederick Patient Patient Cell type: Fibroblasts Cell type: Fibroblasts • HIV * • Lower spare capacity in patients Multiple acyl-CoA dehydrogenase deficiency (MADD) ** Higher spare capacity in patient Data courtesy of Dr. Pinar Coskun,Department of Neurobiology and Behavior at UC Irvine PLoS One. 2009 Dec 17; 4(12):e8329. Song Y et al

15. IMMUNOLOGYSpare Capacity Affects Memory T Cell Development and Survival Naïve T cells Effector T cells Memory T cells Mouse Lymphocytes Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development van der Windt, JW, … and Pearce, EL, Washington School of Medicine. Immunity 2011 December 28

16. CANCERXF Assays Measure Substrate Oxidation Glutamine Fatty Acids Glucose Myc + Myc - OCR value (relative to 25 mM) Osteosarcoma Cells Glucose DLBCLOxPhos cell line panel DBLC non-OxPhos cell line panel MDA-MB-231 Breast Cancer Cells Mitochondrial Complex I Inhibitors and Forced Oxidative Phosphorylation Synergize in Inducing Cancer Cell Death Palorini, et al, International Journal of Cell Biology. 2013(14).  Metabolic changes in cancer cells upon suppression of MYC Anso E, et al., Cancer and Metab; 2013 Feb 4. 1 (1):7 Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma Caro P, et al., Cancer Cell; 2012 Oct 16. 22 (4):547-560.

17. XF Plasma Membrane Permeabilizer (PMP) Measure the biochemistry and mechanisms of mitochondrial dysfunction in intact cells Glu/Mal substrate XF PMP permeabilizes the plasma membrane without damaging mitochondria — healthy control — PT II (Complex I deficient) — PT III (Complex I deficient) Glu/Mal substrate XF PMP Permeabilized Human Skin Fibroblasts Bioenergetics evaluation of intact & permeabilized human skin fibroblasts with mitochondrial disorders using the Seahorse XF96, Invernizzi, et al., European Society for Clinical Investigation Poster, March 22-24, 2012; Budapest, HU

18. XF Palmitate-BSA FAO Substrate Reliable, functional fatty acid oxidation assays are easy with a ready-to-use, bioavailable Palmitate-BSA conjugate HepG2 cells L6 myoblasts

19. Exercise Work in small groups

20. CARDIOVASCULARStress Test Sensitivity Reveals Sub-Lethal Stress Response and Mechanism XF Cell Mito Stress Test FCCP Antimycin A Oligo 10 µM HNE 0 µM HNE 5 µM HNE 20 µM HNE 300 HNE 250 200 OCR (% baseline) 150 Neonatal Rat Ventricular Myocytes 100 50 0 0 25 50 75 100 125 150 175 200 Time (min) Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal Bradford G. Hill, Brian P. Dranka, LuyunZou, John C. Chatham and Victor M. Darley-Usmar, Biochem J. 2009 Nov 15;425(1):99-107

21. CARDIOVASCULARStress Test Sensitivity Reveals Sub-Lethal Stress Response and Mechanism XF Cell Mito Stress Test FCCP Antimycin A Oligo 10 µM HNE 0 µM HNE 5 µM HNE 20 µM HNE 300 HNE 250 200 OCR (% baseline) 150 Neonatal Rat Ventricular Myocytes 100 50 • How does increasing concentrations of HNE affect: • Basal Respiration • ATP Production • Spare capacity • Proton leak 0 0 25 50 75 100 125 150 175 200 Time (min) Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal Bradford G. Hill, Brian P. Dranka, LuyunZou, John C. Chatham and Victor M. Darley-Usmar, Biochem J. 2009 Nov 15;425(1):99-107

22. CARDIOVASCULARStress Test Sensitivity Reveals Sub-Lethal Stress Response and Mechanism XF Cell Mito Stress Test FCCP Antimycin A Oligo 10 µM HNE 0 µM HNE 5 µM HNE 20 µM HNE 300 HNE 250 200 OCR (% baseline) 150 Neonatal Rat Ventricular Myocytes 100 50 0 0 25 50 75 100 125 150 175 200 Time (min) Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal Bradford G. Hill, Brian P. Dranka, LuyunZou, John C. Chatham and Victor M. Darley-Usmar, Biochem J. 2009 Nov 15;425(1):99-107

24. OUT IN e- e- e- e- e- e- e- e- Cyt C Cyt C Cyt C e- e- O2 O2 ATP ADP Q H2O Mitochondrial Electron Transport Chain H+ H+ H+H+H+ H+ H+H+ H+H+ OH- H+H+ H+H+ 2OH- NAD+ H+`H+ H+` NADH Krebs Cycle Peter S. Rabinwitch after Mandavilli et al, Mutation Research 509 (2002) 127–151