XF Glycolysis Stress Test Kit User Manual
Contents
1. ccceeeeeee seen 7 II Optimization ASSA YS cccccc cece ccc ec ec ee cence ee eeeeeneaeaeeeeeeeeneaes 9 IV Running the XF Glycolysis Stress TeSt cccecceeeeeeeee eens 17 V Analyzing XF Glycolysis Stress Test Data cee 20 XF Glycolysis Stress Test Kit User Manual XF24 3 i rse Bioscience Introduction Overview Cellular metabolism is the process of converting fuel substrates such as glucose fatty acids and glutamine to ATP and building blocks through a series of enzymatic oxidation and reduction reactions Cells take up reactants from their environment such as glucose and Os and process them biochemically to generate ATP resulting in the extrusion of products such as lactate H and COs into the extracellular environment ATP produced is used to maintain cellular function and to do work The Seahorse XF Analyzer simultaneously measures these energy producing pathways non invasively in real time through the use of novel solid state sensors specially designed consumables and elegant instrument mechanics The rate of oxygen consumption or OCR is proportional to mitochondrial respiration while the rate of extracellular acidification through the extrusion of protons is proportional to glycolysis Changes in O and H concentrations are measured in a transient microchamber with the solid state sensors residing 200 microns above the cell surface This ability to meas2. Kit User Manual XF24 13 i rse Bioscience Prepare Oligomycin Titrations for Injection Allow vial of DMSO and one aliquot of Oligomycin 5 mM to thaw at room temperature Label and refreeze the Oligomycin aliquot within 1 hour of thaw for subsequent experiments do not discard More than one additional freeze thaw cycle is not recommended 1owB 10 pC 1041D 12 vi DMSO 10ul DMSO 10u DMSO 10u DMSO SNA G suitable dilution vessel according to Figure 5 h aus Create a serial dilution of Oligomycin in microtubes or Oligomycin aliquot SmM Figure 5 Oligomycin Serial Dilution Series Add 7 5 ul of each dilution to 1493 ul of XF Glycolysis Stress Test Optimization Medium Add 80 ul of the resulting solution to injection port A of the appropriate wells following the plate layout shown at right NOTE Injection ports of background wells should be loaded with the highest concentration of Oligomycin however no cells should be seeded in these wells Once the injection ports are loaded gently place cartridge in a CO gt free incubator at 37 C until ready to begin the assay XF Glycolysis Stress Test Kit User Manual XF24 14 ae rse Bioscience Running the Assay with the XF Glycolysis Stress Test Software App 1 Open the XF software Seahorse Bioscience N In the Seahorse Apps drop down menu choose XF Glycolysis Kit S Seahorse Apps Click the Start App button Y Click the R
3. aliquots 1 M NOTE pH adjustment of 2 DG solution may be performed in the reagent bottle or in a separate beaker Before starting ensure the availability of an appropriately sized stir bar 1 Re suspend stock in 16 ml of XF Glycolysis Stress Test Assay Medium pre warmed to 37 C pipetting up and down several times 2 Adjust pH to 7 35 0 05 in stock jar or separate beaker and adjust volume to 18 ml with XF Glycolysis Stress Test Assay Medium NOTE If solution has cooled to below 30C it should be warmed in a water bath prior to pH adjustment Transfer 3 ml to each aliquot vial Store vials not needed immediately at 20 C Record the date of resuspension on the side of the XF Glycolysis Stress Test Kit Thaw a fresh aliquot of Oligomycin and 2 DG for each assay warm to room temperature before use lil Optimization Assays Cell Number Titration Metabolic rates OCR and ECAR can vary significantly between different cell types culturing conditions and experimental treatments Therefore the optimal cell seeding density should be determined on any new cell type before running other optimization or profiling assays For the best results several cell densities should be tested A typical range is between 10 000 and 100 000 cells per well although the actual seeding densities may need to be adjusted depending on cell diameter rate of growth and other factors Cell types that are small in diameter have long doubling times or w
4. before and after the experiment to assess both seeding uniformity and cell morphology Cells which are overly sparse or crowded in the well may exhibit altered metabolic behavior producing a higher degree of variation between wells The optimal cell seeding density should be determined based on the following criteria 1 Minimal ECAR of 20 mpH min 2 Seeding density falls within the linear portion of the graph Visual confirmation of appropriate cell morphology and seeding NOTE As the actual number of cells present in the well will affect the rate of glycolysis observed it is strongly recommended to maintain a consistent number of hours between cell seeding and running the XF assay to minimize variation between experiments XF Glycolysis Stress Test Kit User Manual XF24 12 3 rse Bioscience Oligomycin Titration The optimal concentration of Oligomycin can vary significantly between different cell types culturing conditions and experimental treatments Therefore the optimal Oligomycin concentration should be determined on any new cell type before running the XF Glycolysis Stress Test The Oligomycin Titration should be performed using the optimal cell seeding density as determined in the Cell Number Titration experiment on page 9 Prior to the Day of Assa Cell Seeding Seed cells 24 hours prior to the assay in an XF cell culture microplate and place in a 37 C incubator with the desired level of COs NOTE As the actu
5. on page 7 Perform the Medium Exchange NOTE Medium exchange described here is designed to produce a gt 2 000 fold dilution of the original culture medium 1 Remove all but 50 ul of the culture medium from each well 2 Rinse cells two times with 600 ul of XF Stress Test Glycolysis Optimization Medium ore warmed to 37 C Add 750 ul to each well for a final volume of 800 ul well Place the plate in a 37 C incubator without CO for one hour prior to the assay NOTE The Seahorse XF Prep Station is recommended to ensure accurate final volumes during media exchanges XF Glycolysis Stress Test Kit User Manual XF24 10 3 rse Bioscience Running the Assay with the XF Glycolysis Stress Test Software App 1 Open the XF software Seahorse Bioscience N In the Seahorse Apps drop down menu choose XF Glycolysis Kit srs Seahorse Apps Click the Start App button Y Click the Run Optimization Plate button Select Cell Number Titration from the drop down menu 2 So a Enter the Cell Line Name in the appropriate field Te a XF Glycotysis Stress Test Titration Selection clicking on any well in the column and entering the CESE o Enter the cel number tration for each column by chcking on a w 7 Indicate the cell seeding density for each column by seeding density used Click the Start button Choose a directory to save the file in choose a file name if necessary and click OK 10 Place the cartridge and cali
6. parameters of Glycolysis individually Average Ghynnhytic Glycalyels Glycolytic Capacity Glycolytice Reserve EGAR mpHimin Ewtrarc al liallaar A t hiifeation Extra allhalar A t klieation ECAR impikan E Figure 9 XF Glycolysis Stress Test Results Individual Parameters NOTE If changes are made in the Data Viewer tab the assay must be re exported using the XF Glycolysis Stress Test Software App to obtain the recalculated data output Reference Wu M Neilson A Swift AL Moran R Tamagnine J Parslow D Armistead S Lemire K Orrell J Teich J Chomicz S Ferrick DA Am J Physiol Cell Physiol 2007 Jan 1 292 1 C125 136 XF Glycolysis Stress Test Kit User Manual XF24 21 TE s i rse Bioscience
7. 2995 ul 0 5 uM 4 5 uM 2 7 ul 2997 ul 0 25 uM 2 25 UM 1 35 ul 2999 ul 0 125 uM 1 125 uM 0 675 ul 2999 ul 3 Thawed 2 DG solution is ready to use a Final concentration of 2 DG in the assay will be 100 mM Load Injection Ports Add 80 ul of the prepared compounds into the appropriate injection port and place cartridge in a 37 C incubator without COz Port A Glucose Port B Oligomycin Port C 2 DG _ Gtucose _ Ofigomycin Mij20c _ Media Only XF Glycolysis Stress Test Kit User Manual XF24 18 Perform the Medium Exchange NOTE Medium exchange described here is designed to produce a gt 2 000 fold dilution of the original culture medium 1 Remove all but 50 ul of the culture medium from each well 2 Rinse cells two times with 600 ul of XF Stress Test Glycolysis Assay Medium ore warmed to 37 C Add 510 ul to each well for a final volume of 560 ul well Place the plate in a 37 C incubator without CO for one hour prior to the assay NOTE Using the Seahorse XF Prep Station is recommended to ensure accurate final volumes during media exchanges Running the Assay with the XF Glycolysis Stress Test Software App 1 Open the XF software Seahorse Bioscience 2 Inthe Seahorse Apps drop down menu choose XF Glycolysis Kit seahorse Apps v Tiet Aap Click the Start App button Click the Run XF Glycolysis Stress Test button 5 Enter the information for each group in the appropriate Se Chy
8. XF Glycolysis Stress Test Kit User Manual For use with the XF24 Analyzer Part 102194 100 D2 S iaa aa as AE TAEDE Store at 20 C Al lycolysis Stress test kit For Research Use Only XF Glycolysis Stress Test Kit User Manual XF24 1 T rse Bioscience Preface Copyright 2012 Seahorse Bioscience Inc All rights reserved Printed in U S A Under copyright laws this manual may not be reproduced in any form in whole or in part without prior written permission from Seahorse Bioscience Inc This revision supersedes all previous revisions Every effort has been made to ensure that the information in this manual is accurate at the time of printing However Seahorse Bioscience Inc assumes no liability for errors or omissions and reserves the right to make changes without notice to any products described herein to improve reliability function or design Excel is a registered trademark of Microsoft Corporation Other company and product names may be trademarks of their respective companies Customer Support Phone 800 671 0633 Option 3 Fax 978 671 1611 E mail support seahorsebio com Web http www seahorsebio com Mail Seahorse Bioscience Inc 16 Esquire Road Billerica MA 01862 XF Glycolysis Stress Test Kit User Manual XF24 2 3 rse Bioscience Table of Contents Introduction sore ace ecient ene seeayeateeke sey E AE ceceteeseeeneute 4 ll Preparing the Components of the AsSay
9. al number of cells present in the well will affect the rate of glycolysis observed it is strongly recommended to maintain a consistent number of hours between cell seeding and running the XF assay to reduce variation between experiments For experiments on non adherent cells seeding can be performed immediately prior to the experiment Contact Seahorse Bioscience Support for more details Hydrating the XF Sensor Cartridge The XF Sensor Cartridge must be hydrated prior to the assay e Add 1 0 mL of Seahorse Bioscience Calibrant to each well of an XF Utility Plate e Place the XF Sensor Cartridge on top of the utility plate and place in a 37 C incubator without CO for a minimum of 12 hours Day of Assay Preparation of XF Glycolysis Stress Test Optimization Medium Prepare the XF Glycolysis Stress Test Optimization Medium from the Base Medium as described on page 8 Perform the Medium Exchange NOTE Medium exchange described here is designed to produce a gt 2 000 fold dilution of the original culture medium Remove all but 50 ul of the culture medium from each well Rinse cells two times with 600 ul of Glycolysis Optimization Medium pre warmed to 37 C Add 670 ul to each well for a final volume of 720 ul well pe ae a Place the plate in a 37 C incubator without CO gt for one hour prior to the assay NOTE The Seahorse XF Prep Station is recommended to ensure accurate final volumes during media exchanges XF Glycolysis Stress Test
10. and running the XF assay to reduce variation between experiments NOTE For experiments on non adherent cells seeding can be performed immediately prior to the experiment Contact Seahorse Bioscience Support for more details Hydrating the XF Sensor Cartridge The XF Sensor Cartridge must be hydrated prior to the assay e Add 1 0 mL of Seahorse Bioscience Calibrant to each well of an XF Utility Plate e Place the XF Sensor Cartridge on top of the utility plate and place in a 37 C incubator without CO for a minimum of 12 hours Preparation of XF Glycolysis Stress Test Assay Medium Prepare the XF Glycolysis Stress Test Assay Medium from the Base Medium as described on page 7 Prepare Compounds for Injection Thaw DMSO and one aliquot each of glucose 2 5 M Oligomycin 5mM and 2 DG 1 M at room temperature 1 Add glucose 1 mL to 29 mL of XF Glycolysis Stress Test Assay Medium pre warmed to 37 C Adjust pH to 7 35 0 05 a Final concentration of glucose in the assay will be 10 mM XF Glycolysis Stress Test Kit User Manual XF24 17 3 rse Bioscience 2 Prepare 3 mL of a 9x Oligomycin solution based on the results of the titration performed in Section Ill Optimization Assays on page 9 See table below for easy reference Oligomycin Dose in 9x Solution for Volume of Volume of XF the Assay Injection Oligomycin Stock Glycolysis Stress Test Assay Medium 2 5 uM 22 5 uM 13 5 ul 2987 ul 1 uM 9 uM 5 4 ul
11. bration plate with loaded injection ports on the sliding tray kh piiI for 3 i ee 11 Click Continue to start calibration ET E bene J Tekoop End B Progam Ena 12 When prompted replace the calibration utility plate with the cell plate 13 Click OK then Continue 14 The Optimization Assay will now run on the XF Analyzer 15 When the run is over follow the prompts in the software and remove the cartridge and cell plate and discard une Orecur es AF Gh POOR TEST GPT aaia wane XF Glycolysis Stress Test Kit User Manual XF24 11 ese Biosci Data Analysis and Interpretation The XF Glycolysis Stress Test software output automatically graphs and analyzes the data from the optimization run and offers a recommendation as to the cell seeding density required to achieve a minimum level of ECAR Cell Seeding Density Titration o So N A o o L i ECAR mpH min A 1 Extracellular Acidification N o 1 o 0 20 000 40 000 60 000 80 000 Cell Seeding Density Well Figure 4 Cell Number Titration Optimization Graph In Figure 4 cells were seeded at intervals of 10 000 cells well In this case the seeding density recommended by the software is 20 000 cells well While a particular seeding density may meet the technical requirements of the assay these requirements should be balanced against biological factors Observe the cell plate under a microscope
12. cohrais Sirasa Test Groupa Layean fields and create the plate layout When finished click Next 6 The injection port layout and details are show for reference Click Start button to begin Gtycotyses Test injection Layout Leet Per A 6 ow the assay XF Glycolysis Stress Test Kit User Manual XF24 19 ts Biases 7 Indicate the cell seeding density for each column by clicking on any well in the column and entering the seeding density used 8 Choose a directory to save the file in choose a file name if necessary and click OK 9 Place the cartridge and calibration plate with loaded injection ports on the sliding tray 10 Click Continue to start calibration 11 When prompted replace the calibration utility plate with the cell plate 12 Click Ok then Continue 13 The XF Glycolysis Stress Test will now run on the XF Analyzer 14 When the run is over follow the prompts in the software and remove the cartridge and cell plate and discard V Analyzing the XF Glycolysis Stress Test Data The XF Glycolysis Stress Test Software App delivers a series of automatic calculations including the key parameters of glycolytic function Glycolysis Glycolytic Capacity and Glycolytic Reserve Upon completion of the XF Glycolysis Stress Test a tab is present entitled Glycolysis Stress Test Results in addition to the standard data outputs from an XF Assay Click on this tab to view the calculated me
13. d to as the Glycolytic Capacity of the cell 10mM Glucose Oligomycir 2 DG Glycolytic Reserve 20 Vi Glycolytic Gapacity ECAR mpH min Glycolysis Extracellular Acidification Non glycolytic Acidification 0 10 20 30 40 50 60 70 80 90 100 Figure 1 XF Glycolysis Stress Test Profile This assay provides a standard and comprehensive method to assess the three key parameters of glycolytic function Glycolysis Glycolytic Capacity and Glycolytic Reserve ECAR of cells are first assessed in the absence of extracellular glucose to measure non glycolytic acidification which results from other metabolic pathways occurring in the cells Three different compounds glucose oligomycin and 2 Deoxy D glucose 2 DG are injected consecutively followed by 3 ECAR measurements after each injection The first injection is a saturating concentration of glucose 10 mM Glucose is taken up by the cells and catabolized through the glycolytic pathway to lactate producing ATP and protons The extrusion of protons into the surrounding medium produces a rapid increase in ECAR This glucose induced response is reported as the rate of glycolysis or glycolytic flux under a basal condition The second injection is oligomycin an ATP synthase inhibitor Oligomycin inhibits mitochondrial ATP production and thus shifts the energy production to glycolysis with the subsequent increase in ECAR revealing the maximum glycolytic capacity of the cells The fi
14. erials for XF Glycolysis Stress Test Media o Bicarbonate free low phosphate DMEM Sigma D5030 o NaCl o Phenol Red e g Sigma P0290 o Glucose e g Sigma 8760 o L glutamine e g Invitrogen 25030 ll Preparing the Components of the Assay Preparation of Assay Media There are two types of assay medium utilized in the XF Glycolysis Stress Test Seahorse recommends preparing the following base medium formulated for long term stability and supplementing with additional components immediately prior to running the assay XF Glycolysis Stress Test Base Medium Prepare the Day Prior to the Assay Formulation for 1 L Component Concentration DMEM D 5030 Sigma 1 bottle powder NaCl 143 mM final Phenol Red 3 mg Preparation Instructions 1 Dissolve one bottle of Sigma D5030 powder in 990 mL of TC grade water pre warmed to 37 C Add 1 85 g NaCl for a final concentration of 143 mM Add 0 6 mL of a 0 5 Phenol Red solution Sigma P0290 for a final concentration of 3 mg L Adjust the pH to 7 35 0 05 Sterile filter and store at 4 C It is stable at 4 C for at least 3 months oo oe e ND XF Glycolysis Stress Test Kit User Manual XF24 7 eee rse Bioscience XF Glycolysis Stress Test Optimization Medium Prepare the Day of the Assay Preparation of 100 mL sufficient for one assay plate 1 Place 100 mL of XF Glycolysis Stress Test Base Medium in an appropriate container 2 Add 400 ul of a 2 5 M Glucose solution for a fi
15. hich are known to be less metabolically active may require higher seeding densities to achieve optimal results An example plate layout diagram is provided below X 20k 30k 40k 50k 60k 10k 20k 30k X 50k 60k 10k 20k X 40k 50k 60k 10k 20k 30k 40k 50k X NOTE X wells should contain only Optimization Medium no cells XF Glycolysis Stress Test Kit User Manual XF24 9 i rse Bioscience Prior to the Day of Assa Cell Seeding Seed cells 24 hours prior to the assay in an XF cell culture microplate and place in a 37 C incubator with the desired level of COs NOTE As the actual number of cells present in the well will affect the rate of glycolysis observed it is strongly recommended to maintain a consistent number of hours between cell seeding and running the XF assay to reduce variation between experiments NOTE For experiments on non adherent cells seeding can be performed immediately prior to the experiment Contact Seahorse Bioscience Support for more details Hydrating the XF Sensor Cartridge The XF Sensor Cartridge must be hydrated prior to the assay e Add 1 0 mL of Seahorse Bioscience Calibrant to each well of an XF Utility Plate e Place the XF Sensor Cartridge on top of the utility plate and place in a 37 C incubator without CO for a minimum of 12 hours Preparation of XF Glycolysis Stress Test Optimization Medium Prepare the XF Glycolysis Stress Test Optimization Medium from the Assay Base Medium as described
16. nal concentration of 10 mM 3 Add 1mL of a 200 mM L glutamine solution for a final concentration of 2 mM 4 Warm to 37 C and adjust the pH to 7 35 0 05 XF Glycolysis Stress Test Assay Medium Prepare the Day of the Assay Preparation of 100 mL sufficient for one assay plate 1 Place 100 mL of XF Glycolysis Stress Test Base Medium in an appropriate container 2 Add 1mL of a 200 mM L glutamine solution for a final concentration of 2 mM 3 Warm to 37 C and adjust the pH to 7 35 4 0 05 Reagent Preparation NOTE Wear chemically resistant impervious gloves when handling the compounds in the kit Reagent preparation must be performed in a Class Il Biological Safety Cabinet Reagent aliquots must be stored at 20 C Use reagent aliquots within 3 months of reconstitution Remove the XF Glycolysis Stress Test Kit from the freezer and allow the Oligomycin and 2 DG stock vials to warm to room temperature Store the remaining components in the freezer until the reagent preparations are complete Preparation of Oligomycin Aliquots 5 mM 1 Spin down the stock vial in a micro centrifuge for approximately 5 seconds prior to opening 2 Resuspend powder in 180 ul of DMSO 3 Transfer 30 ul to each aliquot vial Store vials not needed immediately at 20 C 4 Record the date of resuspension on the side of the XF Glycolysis Stress Test Kit XF Glycolysis Stress Test Kit User Manual XF24 8 i rse Bioscience Preparation of 2 Deoxy D Glucose
17. nal injection is 2 DG a glucose analog which inhibits glycolysis through competitive binding to glucose hexokinase the first enzyme in the glycolytic pathway The resulting decrease in ECAR further confirms that the ECAR produced in the experiment is due to glycolysis The difference between Glycolytic Capacity and Glycolysis rate defines Glycolytic Reserve XF Glycolysis Stress Test Kit User Manual XF24 5 i rse Bioscience Optimization Glycolysis Stress Test Automated Data e Cell Number Titration Output amp Metrics e Oligomycin Titration Figure 2 General workflow of the XF Glycolysis Stress Test Kit XF Glycolysis Stress Test Kit Contents The XF Glycolysis Stress Test Kit contains enough reagents to run 6 full XF assay plates Glucose Solution Oligomycin Figure 3 XF Glycolysis Stress Test Kit Contents Reagent Quantity Seahorse Glucose Solution 6 vials of a 2 5 M solution Seahorse Oligomycin 1 vial of powder 6 empty vials for reconstituted aliquots Seahorse 2 DG 1 vial of powder 6 empty vials for reconstituted aliquots Seahorse DMSO 1 vial of 1 mL Materials Required Not provided in the kit e XF24 FluxPak Seahorse Bioscience Cat No 100850 001 e Serial dilution vessel 24 well plate or 1 5 mL tubes e CO free incubator set to 37 C e XF Glycolysis Stress Test recommended assay medium as described on page 7 XF Glycolysis Stress Test Kit User Manual XF24 6 Oe rse Bioscience e Mat
18. t co qa O SE 5 x 5E te Sg SO BS x Wi N o o 0 00 0 13 0 25 0 50 1 00 2 50 0 13 0 25 0 50 1 00 2 50 Oligomycin Concentration uM Oligomycin Concentration uM Figure 6 Oligomycin Titration Optimization Result Graphs In Figure 6 the XF Glycolysis Stress Test software would recommend a concentration of 0 5 uM The optimal Oligomycin concentration should be determined based on the following criteria 1 Maximum inhibition of OCR as shown by the plateau in the dose response curve 2 Maximum ECAR lt 120 mpH min If the maximum ECAR value exceeds 120 mpH min a lower cell seeding density should be selected and the Oligomycin Titration should be repeated at this cell seeding density NOTE As the actual number of cells present in the well will affect the rate of glycolysis observed it is strongly recommended to maintain a consistent number of hours between cell seeding and running the XF assay to minimize variation between experiments XF Glycolysis Stress Test Kit User Manual XF24 16 3 rse Bioscience IV Running the XF Glycolysis Stress Test Prior to the Day of Assay Cell Seeding Seed cells 24 hours prior to the assay in an XF cell culture microplate and place in a 37 C incubator with the desired level of COs NOTE As the actual number of cells present in the well will affect the rate of glycolysis observed it is strongly recommended to maintain a consistent number of hours between cell seeding
19. trics from the experiment XF Glycolysis Stress Test Kit User Manual XF24 20 a 2 di CER Seahorse Bioscience a l Ghycolysis Stress Test Results Dais way Ghreslysis Stress Test EA i i uo i ww at ie T u 2S S 4 i ie al Ay ABT nig HY 4 F a wm a j li P p EiT E H H ih rs a 4 4 wit 2 3 4 8 6 oF 8 8 ono J i 3 Bote Hr of ECAR Tle ji Pec For Cot CL Ge ep Kay Parameters of Glycalysis Harmi AA A oa ai a AY Soap er others Geter bo Wee Ft Were Por de ee x iy 3 mi p rs t Le i ii i i of i i i I e eaha Capactipyecayne Heserye ey a e e i Er tha as te Gata Ac iihi thii ECAR mpallrnin Figure 7 XF Glycolysis Stress Test Summary Results Kinetic Results This graph top left shows the average ECAR and standard deviation data obtained at each measurement point for all groups in a graphical form This data presentation highlights the dynamic changes within and between groups during the experiment Data Table The numerical results from the kinetic graph are presented in tabular form to simplify data conversion export for additional analyses Histogram This graph bottom left shows the average ECAR and standard deviation data for the three key parameters of Glycolysis for all groups In addition to these summary outputs the XF Glycolysis Stress Test Software App also produces tabular and graphical presentations of the three key
20. un Optimization Plate button Select Oligo Titration from the drop down menu Enter the Cell Line Name in the appropriate field e a Oe oS Enter the cell seeding number in the appropriate field NOTE If desired the range of Oligomycin concentrations may be adjusted In this case the serial dilution volumes must also be adjusted accordingly Click the Start button Choose a directory to save the file in choose a file name if necessary and click OK 10 Place the cartridge and calibration plate with di 6 1 23Measwe for 3 Min 0 sec O 7 Loop End loaded injection ports on the sliding tray 11 Click Continue to start calibration 12 When prompted replace the calibration utility plate with the cell plate 13 Click Ok then Continue Assn lara 14 The Optimization Assay will now run on the XF Analyzer 7 15 When the run is over follow the prompts in the software and remove the cartridge and cell plate and discard XF Glycolysis Stress Test Kit User Manual XF24 15 ts Biases Data Analysis and Interpretation The XF Glycolysis Stress Test software output automatically graphs and analyzes the data from the optimization run and offers a recommendation as to the optimal Oligomycin concentration to use in the XF Glycolysis Stress Test Oligomycin Titration OCR Rate 4 Oligomycin Titration ECAR Rate 7 _ N o l So So o oa o A o c raj S ST of 2c ZE ES 3E J ng o
21. ure cellular metabolism has enabled scientists worldwide to advance their understanding of the roles glycolysis and mitochondrial respiration play in human physiology and disease Changes in cellular metabolism have been shown to have therapeutic and diagnostic potential in the areas of obesity diabetes aging cancer cardiovascular function immunology and more recently translational medicine Assay Principle Glycolysis and oxidative phosphorylation are the two major energy producing pathways in the cell Most cells possess the ability to shift dynamically between these two processes adapting metabolically to changes in their environment for the purpose of survival Glucose in the cell is converted to pyruvate which is then converted to lactate in the cytoplasm or CO2 and water in the mitochondria Glucose conversion to lactate Glycolysis results in a net production and extrusion of protons into the extracellular medium As glycolysis occurs the resulting acidification of the medium surrounding the cells is measured directly by the XF Analyzer and reported as the Extracellular Acidification Rate or ECAR XF Glycolysis Stress Test Kit User Manual XF24 4 3 rse Bioscience When oxidative phosphorylation is disrupted by an ATP synthase inhibitor Oligomycin cells may increase the rate of glycolysis maintain ATP production and thereby energy homeostasis as previously shown Wu M et al This elevated rate of glycolysis is referre
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