XF Glycolysis Stress Test Kit User Manual
Contents
1. ccc ccc ene e ence cease este eens eneneeeaeeeaneeeas 9 IV Running the XF Glycolysis Stress Test cccseeeeeeeeee eens 17 V Analyzing XF Glycolysis Stress Test Data ccecee eens 21 XF Glycolysis Stress Test Kit User Manual XF96 3 i j Seahorse Bioscience Introduction Overview Cellular metabolism is the process of converting fuel substrates such as glucose fatty acids and glutamine to ATP through a series of enzymatic oxidation and reduction reactions Cells take up reactants from their environment such as glucose and Oz and process them biologically to generate ATP resulting in the extrusion of products such as lactate H and CO 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 toglycolysis Changes in O2 and H concentrations are measured in a transient microchamber with the solid state sensors residing 200 microns above the cell surface This ability to measure cellular metabolism has enabled scientists worldwide to advance their un2. 1 5 ml 1 5ml 1 5ml 1 5ml 1 5 ml 1 5ml 7 DMEM DMEM DMEM DMEM DMEM DMEM DMEM DMEM DMEM D D aadd j Use as is to load injection ports 5uM 2 5puM 1 0 uM 5 uM 25 uM 125uM 0625uM 0312uM 0156 uM 0 uM Oligomycin concentration after injection into assay N t o i Figure 5 Oligomycin Serial Dilution Series NOTE 5 0uM and 2 5uM aliquots are prepared directly from oligomycin stock vial in DMEM Other aliquots are prepared via a 2 step dilution first in DMSO followed by DMEM The concentration of DMSO after injection into the assay is 0 05 0 1 in 5 uM Add 25ul 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 free incubator at 37 C until ready to begin the assay XF Glycolysis Stress Test Kit User Manual XF96 14 Seahorse Bioscience Running the Assay with the XF Glycolysis Stress Test Software App 1 Open the XF software In the Seahorse Apps drop down menu choose XF Glycolysis Kit EPD Seahorse Apps Click the Start App button Click the Run Optimization Plate button N biai Ap Select Oligo Titration from the drop down menu Enter the Cell Line Name in the appropriate field
3. 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 200ul 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 Glycolysis Stress Test Optimization Medium Prepare the Glycolysis Stress Test Optimization Medium from the Assay Base Medium as described on page Perform the Medium Exchange NOTE Medium exchange described here produces a gt 1000 fold fold dilution of the original culture medium This ts the minimum recommended dilution to minimize effects on the assay from residual growth medium 1 Add 225uL Stress Test Glycolysis Optimization Medium pre warmed to 37 C Remove all but 25ul of the culture medium from each well Rinse cells two times with 300ul of Stress Test Glycolysis Optimization Medium Add 225ul to each well for a final volume of 250ul well g amp N 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 XF96 10 i Seahorse Bioscience Running the Assay with the XF Glycolysis Stress Test Software App 1 Open the XF software 2 Inthe Seahorse Apps drop d
4. Cell Seeding Density Well Figure 4 Cell Number Titration Optimization Graph prepared on XF24 Analyzer In Figure 4 cells were seeded at intervals of 10 000 cells well in a 24 well microplate on an XF24 Analyzer 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 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 Minimum ECAR of 20mpH min 2 Seeding density falls within the linear portion of the graph 3 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 XF96 12 i i Seahorse Bioscience Oligomycin Titration The optimal concentration of Oligomycin can vary significantly between different cell types culturing conditions and experi
5. XF Glycolysis Stress Test Kit Preparation of 2 Deoxy D Glucose aliquots 1M 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 16ml 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 18ml with Glycolysis Stress Test Assay Medium NOTE If solution has cooled to below 30 C it should be warmed in a water bath prior to pH adjustment Transfer 3ml to each aliquot vial Store vials not needed immediately at 20 C Record the date of re suspension 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 XF Glycolysis Stress Test Kit User Manual XF96 8 i j Seahorse Bioscience lll 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 well although the actual seeding densities may need to b
6. XF Glycolysis Stress Test recommended assay medium as described on page 7 e Materials for Glycolysis Stress Test Media o Bicarbonate free low phosphate DMEM Sigma D5030 NaCl Phenol Red e g Sigma P0290 Glucose e g Sigma G8769 L glutamine e g Invitrogen 25030 O O O O XF Glycolysis Stress Test Kit User Manual XF96 6 4 Seahorse Bioscience 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 Glycolysis Stress Test Base Medium Prepare the Day Prior to the Assay Formulation for 1L Component Concentration DMEM D 5030 Sigma 1 bottle powder NaCl 143mM final Phenol Red 3mg Preparation Instructions 1 Dissolve one bottle of Sigma D5030 powder in 990mL of TC grade water pre warmed to 37 C Add 1 85g NaCl for a final concentration of 143mM Add 0 6mL of a 0 5 Phenol Red solution Sigma P0290 for a final concentration of 3mg 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 Ye Glycolysis Stress Test Optimization Medium Prepare the Day of the Assay Preparation of 150mL sufficient for one assay plate 1 Place 150mL of Glycolysis Stress Test Base Medium in an
7. a o a a oe 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 Son 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 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 XF Glycolysis Stress Test Kit User Manual XF96 15 m h Ri i 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 7 120 100 s c ET ST ag 80 EE E D ke a 2 oz 52 604 lt Os 5s Sg ao 28 40 3 O e 20 x 0 0 00 0 13 0 25 0 50 1 00 2 50 0 13 0 25 0 50 1 00 Oligomycin Concentration uM Oligomycin Concentration uM Figure 6 Oligomycin Titration Optimization Resul
8. XF Glycolysis Stress Test Kit User Manual For use with the XF96 Analyzer Part 102194 100 For Research Use Only XF Glycolysis Stress Test Kit User Manual XF96 1 4 4 Seaho 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 1 978 671 1600 Option 3 Toll free within USA 800 671 0633 Option 3 Fax 1 978 671 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 XF96 2 i j Seahorse Bioscience Table of Contents l Introduction E E E aneneauesaoes 4 ll Preparing the Components of the AsSay cceceeeeneeenees 7 Il Optimization ASSAYS
9. appropriate container 2 Add 600ul of a 2 5M Glucose solution for a final concentration of 10mM 3 Add 1 5mL of a 200mM L glutamine solution for a final concentration of 2mM 4 Warm to 37 C and adjust the pH to 7 35 0 05 Glycolysis Stress Test Assay Medium Prepare the Day of the Assay Preparation of 150mL sufficient for one assay plate 1 Place 150mL of Glycolysis Stress Test Base Medium in an appropriate container 2 Add 1 5mL of a 200mM L glutamine solution for a final concentration of 2mM 3 Warm to 37 C and adjust the pH to 7 35 0 05 XF Glycolysis Stress Test Kit User Manual XF96 7 i i Seahorse Bioscience 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 5mM 1 Spin down the stock vial in a micro centrifuge for approximately 5 seconds prior to opening 2 Re suspend powder in 18ul of DMSO 3 Transfer 30ul to each aliquot vial Store vials not needed immediately at 20 C 4 Record the date of re suspension on the side of the
10. derstanding 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 CO 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 XF96 4 i i Seahorse Bioscience When oxidative phosphorylation is disrupted by an ATP synthase inhibitor Oligomycin cells may increase the rate of glycolysis to maintain ATP production and thereby energy homeostasis as previously shown Wu M et a This elevated rate of glycolysis is referred to as the Glycolytic Capacity of the cell 10mM Glucose Oligo
11. e adjusted depending on cell diameter rate of growth and other factors Cell types that are small in diameter have long doubling times or which are known to be less metabolically active may require higher seeding densities to achieve optimal results An example plate layout diagram for a single cell line is provided below 10k 10k 10k 10k 10k 10k 10k 10k X KX KK KK K XK XF Glycolysis Stress Test Kit User Manual XF96 20k 20k 20k 20k 20k 20k 20k 20k 30k 30k 30k 30k 30k 30k 30k 30k 40k 40k 40k 40k 40k 40k 40k 40k 50k 50k 50k 50k 50k 50k 50k 50k 60k 60k 60k 60k 60k 60k 60k 60k 70k 70k 70k 70k 70k 70k 70k 70k 80k 80k 80k 80k 80k 80k 80k 80k NOTE X wells should contain only Optimization Medium no cells 90k 90k 90k 90k 90k 90k 90k 90k 100k 100k 100k 100k 100k 100k 100k 100k x KX KK KK K XK Seahorse Bioscienc 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 CO gt 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
12. eding 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 200ul 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 Glycolysis Stress Test Assay Medium Prepare the 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 0M Oligomycin S5mM and 2 DG 1M at room temperature 1 Add glucose 1mL to 29mL 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 10mM XF Glycolysis Stress Test Kit User Manual XF96 17 i i Seahorse Bioscience 2 Prepare 3mL of a 9x Oligomycin solution based on the results of the titration performed in Section III Optimization Assays on page 9 See table below for easy reference Oligomycin Dose 9x Solution for Volume of in the Assay uM Injection uM Oligomycin Stock pl Volume of XF Glycolysis Stress 5 45 2 5 22 5 1 9 0 5 4 5 0 25 2 25 0 125 1 125 3 Thawed 2 DG solution is ready to use a Final concentration of 2 DG in the assay will be 100mM Load Injection Po
13. 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 metrics from the experiment Glycolysis Stress Test Results Date 6 8 12 Glycolysis Stress Test C2C12 GST SAGE GST C2012 GST ECAR 90 SFI88F GST Measurement regi Leni oe 2 ee Measurements Note Normalization of ECAR measurements is recommended for comparing experimental groups Key Parameters of Glycolysis Normalization can be automated with the XF Analyzer Software Refer to the Kit Manual for details c2c12GsT mSF188F GST o O O Figure 7 XF Glycolysis Stress Test Summary Results t Extracellular Acidification ECAR mpHimin Glycolysis Glycolytic Glycolytic Reserve Capacity Kinetic Results Figure 7 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 XF Glycolysis Stress Test Kit User Manual XF24 21 j Seahorse Bioscience Histogram This shows the average ECAR and
14. lycolysis Stress Test button 5 Enter the information for each group in the appropriate 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 Ghooyeis Test Ingecton Layout the assay Fr fF FF FF iF oF E F sF E F F P F F F E XF Glycolysis Stress Test Kit User Manual XF96 19 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 XF Glycolysis Stress Test Kit User Manual XF24 20 LC TEAT AHE Eperme is Renning Prona piara p i Cabinas Probes Jo Equilibria dome GL a 2 2 olin for F lin O ga B Tina Deisy ig hy az al Se jiji Te E 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
15. mental 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 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 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 200ul 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 Glycolysis Stress Test Optimization Medium Prepare the Glycolysis Stress Test Optimization Medium from the Base Medium as described on page 8 Perform the Medium Exchange NOTE Medium exchange described here produces a gt 1000 fold fold dil
16. myci 2 DC Glycolytic Reserve 20 Glycolytic Capacity 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 Non glycolytic acidification which include includes CO evolution followed by its hydration to carbonic acid and bicarbonate and proton extrusion are also reported in the same assay Initially cells are incubated in the glycolysis stress test medium without glucose and ECAR is assessed The first injection is a saturating concentration of glucose 10mM 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 basal conditions 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 final injection is 2 DG a glucose analog which inhibits glycolysis through competitive binding to glucose hexoki
17. nase 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 but it is not required for calculating Glycolytic Flux or Glycolytic Capacity The difference between Glycolytic Capacity and Glycolysis rate defines Glycolytic Reserve ECAR prior to glucose injection is referred to non glycolytic acidification which includes CO evolution from the TCA cycle and other biochemical pathways followed by its hydration to carbonic acid and bicarbonate as well as proton extrusion XF Glycolysis Stress Test Kit User Manual XF96 5 r Seahorse 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 Figure 3 XF Glycolysis Stress Test Kit Reagent Quantity Seahorse Glucose Solution 6 vials of a 2 5M 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 1mL Materials Required not provided in the kit e XF96 4 port FluxPak e Serial dilution vessel 24 well plate or 1 5mL tubes e CO free incubator set to 37 C e
18. own menu choose XF Glycolysis Kit Click the Start App button Click the Run Optimization Plate button Select Cell Number Titration for one or two cell lines from the drop down menu 6 Enter the Cell Line Name in the appropriate field 7 Indicate the cell seeding density for each column by clicking on any well in the column and entering the 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 calibration plate with 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 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 XF Glycolysis Stress Test Kit User Manual XF96 11 Seahorse Bioscience daedesd Seahorse Apps Ca Tiersen Selection E 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 So So 80 4 60 4 40 4 Extracellular Acidification ECAR mpH min 20 4 0 20 000 40 000 60 000 80 000
19. rts 27 13 5 5 4 2 1 1 35 0 675 Test Assay Medium ul 2973 2987 2995 2997 2999 2999 Add 25ul of the prepared compounds into the appropriate injection port and place cartridge in a 37 C incubator without CO EF Giycolyeis Sireasa Tori Giycolysis Tesi Injacton Layout XF Glycolysis Stress Test Kit User Manual XF96 18 Port A Glucose Port B Oligomycin Port C 2 DG Perform the Medium Exchange NOTE Medium exchange described here produces a gt 1000 fold fold dilution of the original culture medium This is the minimum recommended dilution to minimize effects on the assay from residual buffer capacity of growth medium 1 Add 225uL Stress Test Glycolysis Optimization Medium pre warmed to 37 C Remove all but 25ul of the culture medium from each well Rinse cells two times with 300ul of Stress Test Glycolysis Assay Medium pre warmed to 37 C Add 150ul to each well for a final volume of 175ul well a eS 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 meini Seahorse Apps i Click the Start App button XF Giycotysis Kit y ibt Lsp Click the Run XF G
20. 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 parameters of Glycolysis individually Figure 8 below D LAD 3 CC i CSTE F Glycolysis Extraceiu er Acidification ECAR mpHinin ao C2C12 GST SFI88F GST Extracellular Acidification 0 apaG y C2C12 GST i Co Ty E ECAR mptimnin Average Glycolytic Capacity eo s0 a 40 E 7 20 F 10 5 C2C12 GST SF186F GST Figure 8 XF Glycolysis Stress Test Results Individual Parameters on B NESAS pupi Reserve C2C12 GST M a E ey E Glycolytic Reserve C2C12 GST SF186F GST 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 References 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 lbrahim Hashim A et al Free Base Lysine Increases Survival and Reduces Metastasis in Prostate Cancer Model J Cancer Sci Ther 2011 XF Glycolysis Stress Test Kit User Manual XF24 22 Seahorse Biosciena
21. t Graphs prepared on an XF24 Analyzer In Figure 6 the XF Glycolysis Stress Test software would recommend a concentration of 0 5 uM This example titration assay was conducted in a 24 well microplate on an XF24 Analyzer 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 90mpH min If the maximum ECAR value exceeds 90mpH 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 XF96 16 i Seahorse Bioscience IV Running the XF Glycolysis Stress Test 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 CO 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 se
22. ution of the original culture medium This is the minimum recommended dilution to minimize effects on the assay from residual buffer capacity of growth medium Add 225uL Stress Test Glycolysis Optimization Medium pre warmed to 37 C Remove all but 25ul of the culture medium from each well 1 2 3 Rinse cells two times with 300ul of Glycolysis Optimization Medium pre warmed to 37 C 4 Add 200ul to each well for a final volume of 225ul well 5 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 XF96 13 i Seahorse Bioscience Prepare Oligomycin Titrations for Injection Allow vial of DMSO and one aliquot of Oligomycin 5mM 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 Prepare dilutions from oligomycin stock vial according to the diagram below 8ul A 10ul B 10ul C 10pI D 10pI E 10uI F 10pl G 12 ul 10ul 10 10 10ul 10ul 10ul 10ul DMSO DMSO DMSO DMSO DMSO DMSO DMSO EN AANA Nag h roe ENGNG NG Ea f D Hy MO Oligomycin l 5mM cu J l H v b v v 7 5 ul A 7 5 ul m 7 5 ul C 7 5 ul D 5 ul E 5 ul F v ul l 5 ul H B ul I 0 ml 1 5 ml 1 5 ml eat
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