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1. MitoXpress Xtra Oxygen Consumption Assay HS Method tr _ t pdt For the measurement of Extracellular Oxygen Consumption E E ary V EM dS x S E eo x ILLUMINATING DISCOVERY P L MitoXpress Xtra Oxygen Consumption Assay HS Method For the measurement of Extracellular Oxygen Consumption For use with e Adherent cells e Suspension cells e Permeabilised cells e Isolated mitochondria e 3D cultures tissues spheroids e Raft and scaffolds e Isolated enzymes e Bacteria yeasts and moulds TABLE OF CONTENTS GENERAL INFORMATION e 3 MATERIAL SUPPLIED jasa saa pasa aapa nagan a Dena aji paragan e attese 3 STORAGE AND STABILI Y uuu u uu eae 3 ADDITIONAL ITEMS REQUIRED U U uu uu etes 3 OPTIONAL TEMS NOT SUPPLIED J mte nente 3 DESCRIPTION ttt 4 FLOW DIAGRAM 5 PLATE READER SET DP cee cere u R u u 6 MEASUREMENT PARAMETERS nee 6 INSTRUMENTS AND SETTINGS ull ade u asoa 6 SIGNAL OPTIMISATION u LLL u L anandangana reet 7 PERFORMING THE OXYGEN CONSUMPTION ASSAY 8 CELL CULTURE AND PLATING u u u u u u aa mataanan naa aana 8 PRE ASSAY PREPARATION J tette btt 8 TYPICAL ASSAY Lesen tatit Bae aknas sma Qanun tutem 9 ANAE SIS soc ee eee ore a ang ah gagana 11 ASSESSING OXY GEN CONSUMPTION u u u u u u u u uuu uu 11 PLOTTING ADUSE RESPONSE CURVE aaa uuu aaah 12 CELLULAR ENERGY FLUX ANALYSIS tette 13 APPENDIX A INS2. P15 RECOMMENDED INSTRUMENT AND MEASUREMENT SETTINGS Instrument Optical Configuration Integration 1 Da W1 Integration 2 D2 W2 Ex nm Em nm BMG Labtech FLUOStar Omega POLARstar Omega CLARIOstar Filter based Top or bottom read 30 30ys 70 30ys Dual read TR F Lifetime Ex 340 50nm TR EX L Em 650 x 50nm BP 655 BMG Labtech PHERAstar FS Filter based Top read 40 100us n a Ex 337nm HTRF Module Em 665nm HTRF Module BMG Labtech FLUOStar Optima POLARstar Optima Filter based Top or bottom read 30 100us n a Ex 340 50nm TR EX L Em 655 x 50nm BP 655 Perkin Elmer VICTOR series X4 X5 Filter based Top read 30 30ys 70 30ys Dual read TR F Lifetime Ex 340 40nm D340 Em 642 10nm D642 Perkin Elmer EnVision EnSpire Filter based Top read 40 100us n a TR F Ex 340nm 60nm X340 Em 650nm 8nm M650 BioTek Synergy H1 H4 HT 2 Cytation 3 Filter based Top or bottom read 30 30s 70 30ys Dual read TR F Lifetime Ex 380 20nm Em 645 15nm BioTek Mx H1m Monochromator Filter based Top or bottom read 30 100us n a TR F Ex 380 20nm Em 650 15nm Tecan Infinite Safire Genios Pro Monochromator Filter based Top or bottom read 30 100us n a Ex 380 20nm Em 650 20nm Mol Devices SpectraMax Flexstati
3. TR F focal height Repeat without phenol red or serum Repeat as top or bottom read respectively Increase volume of MitoXpress Xtra 15ul Contact Instrument Supplier for further options ON UT Q N FREQUENTLY ASKED QUESTIONS Q What do l do if cannot detect any signal in wells containing cells and MitoXpress Xtra or can detect a signal but the slope rate appears very low A Check correct Instrument Settings Appendix A Perform Signal Optimisation Include GOx control max signal Increase cell density If tested and not resolved contact TechSupport luxcel com Q What do do if can detect a signal in wells containing cells and MitoXpress Xtra but the slope rate falls initially or is variable from well to well A Check cell seeding and pipetting consistency Increase cell density Ensure plate instrument and all culture media and stock solutions are pre warmed at 37 C prior to use Reduce plate preparation times NOTE Some plate readers have inconsistent temperature control If you suspect this to be the case consider Reduce assay and equilibration temperatures to 30 C and avoid outer wells If tested and not resolved contact TechSupport luxcel com P19 P20 REFERENCES Prediction of liver injury induced by chemicals in human with a multiparametric assay on isolated mouse liver mitochondria Porceddu M et al Toxicol Sci 2012 Oct 129 2 332 45 A high throughput dual parame
4. 0yl of MitoXpress Xtra in media stock to each well Figure 4 Add 150yl of fresh culture media only no MitoXpress Xtra to the Blank Control wells STEP 3 Test compound stock or vehicle typically 1 10pl may be added at this point if desired NOTE We recommend keeping the volume of added compound low to minimise any potential effects of solvent vehicle Figure 4 Aliquoting fresh media MitoXpress Xtra P9 STEP 4 Promptly seal each well by adding two drops or 100yl pre warmed HS Mineral Oil ae care to avoid air bubbles Figure 5 NOTE Small variations in the volume of oil between 90 110ul should not adversely affect the readings using MitoXpress Xtra See also Appendix B HS Mineral Oil Pipetting Tips STEP 5 Read the plate immediately in a fluorescence plate reader with the set up as described in Appendix A Instrument Settings Figure 6 The plate should be measured kinetically for gt 90 minutes When measurement is completed remove the plate and save measured data to file Optional Controls e Signal Controls Leave 2 or 3 wells free from the addition of cells for use as Signal Controls Add 150ul of fresh culture media 10pl of reconstituted MitoXpress Xtra reagent to each well P e Positive Controls Leave 2 or 3 wells free from the addition of cells for use as Positive Controls Add 150ul of fresh culture media 10yl of 1mg ml Glucose Oxidase stock solution in water 10ul recon
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6. TRUMENT SETTINGS aaa 14 APPENDIX B HS MINERAL OIL PIPETTING TIPS 17 APPENDIX C TROUBLE SHOOTING aaa 18 REFERENCE onem MM E 20 RELATED PRODUCTS u eu 21 GENERAL INFORMATION MATERIALS SUPPLIED Assay kit will arrive at room temperature For best results store as indicated below Item 96 well Quantity Size Storage MitoXpress Xtra reagent 1 vial 4 C HS 100D HS Mineral Oil 1 dropper bottle 15ml Room Temp dark STORAGE AND STABILITY The MitoXpress Xtra reagent should be stored as follows e Dry material between 2 to 8 C see Use Before date on vial e Reconstituted product can be aliquoted at 20 C Use within one month avoid freeze thaw ADDITIONAL ITEMS REQUIRED e Fluorescence plate reader with suitable filter and plate temperature control e 96 well black wall clear bottom TC plates or standard PS plates for cell culture OPTIONAL ITEMS NOT SUPPLIED e Repeater pipette recommended e Plate block heater for plate preparation SUPPORT e Visit our website www luxcel com k May also be used in a 384 well format with one vial of probe sufficient for 200 wells P3 PA DESCRIPTION Luxcel s MitoXpress Xtra Oxygen Consumption Assay HS Method is a highly flexible 96 or 384 well fluorescence plate reader based approach for the direct real time analysis of cellular respiration and mitochondrial function The easy to use MitoXpress Xtra assay allows measuremen
7. atiometric TR F measurement Lifetime calculation NOTE Further details including instrument filter selection and measurement settings can be found in Appendix A Instrument Settings SIGNAL OPTIMISATION recommended for first time users NOTE Use a plate block heater for plate preparation and pre warm plate reader to measurement temperature STEP 1 Reconstitute contents of the MitoXpress Xtra vial in 1ml of water PBS or culture media gently aspirating 3 4 times NOTE Reconstituted probe stock can be stored in the dark between 2 to 8 C for several days or stored as aliquots in water at 20 C for use within one month avoid freeze thaw STEP 2 Prepare 8 replicate wells of a 96 well plate by adding 150pl pre warmed culture medium to each well A1 A4 B1 B4 STEP 3 Add 10yl reconstituted MitoXpress Xtra reagent to 4 of the replicate wells A1 A4 and 10yl water PBS or media to the remaining replicates wells B1 B4 STEP 4 Promptly add two drops or 100ul pre warmed HS Mineral Oil to all eight replicate wells taking care to avoid air bubbles NOTE See Appendix B HS Mineral Oil Pipetting Tips STEP 5 Read plate immediately in a fluorescence plate reader over 30 minutes read every 2 3 minutes STEP 6 Examine Signal Control well A1 A4 and Blank Control well B1 B4 readings linear phase and calculate S B ratio NOTE For dual read TR F calculate S B for each measurement window For most fluorescence plate reade
8. d for the Signal Control sample without cells should be subtracted from all test values P11 Data analysis templates are available from some plate reader manufacturers specifically configured to automate the analysis of Luxcel s MitoXpress range of assays Microsoft Excel templates are also available through our website www luxcel com PLOTTING A DOSE RESPONSE CURVE To generate a dose response curve plot the data generated as outlined above against the corresponding compound concentration Figure 8 Antimycin A 3 co Q o 0 1 1 Drug Concentration uM Figure 8 The dose response curve presented here is an example of the data typically produced with this assay Drug concentration uM versus calculated slope us hour demonstrates that this drug causes inhibitory response on cellular respiration P12 CELLULAR ENERGY FLUX ANALYSIS Multiparametric or multiplex combination of MitoXpress Xtra Oxygen Consumption Assay HS Method together with Luxcel s pH Xtra Glycolysis Assay Cat No PH 100 allows the simultaneous real time measurement of mitochondrial respiration and glycolysis and analysis of the metabolic phenotype of cells and the shift flux between the two pathways under pathological states Figure 9 E ate Consumption J Glycolysis n Rotenone Antimycin FCCP Oxamate Untreated Figure 9 Cellular Energy Flux for HepG2 cells treated with a combination of drug compounds mo
9. dulating the ETC or inhibiting lactate production shown as a percentage relative to untreated control cells Comparative measurements with MitoXpress Xtra and pH Xtra show the shift between mitochondrial respiration and glycolysis and the cellular control of energy ATP measured 1h post treatment using Promega Cell Titer Glo P13 P14 APPENDIX A INSTRUMENT SETTINGS Three fluorescence modalities can be successfully used depending on plate reader type and instrument setup NOTE We strongly recommend only using fluorescence plate readers equipped with temperature control Basic Intensity Measurement Measurement of signal Intensity sometimes referred to as Prompt provides flexibility to use a very wide range of commonly available fluorescence monochromator or filter based plate readers Optimal wavelengths are 380nm excitation and 650nm for emission with detection Gain parameters PMT typically set at medium or high NOTE MitoXpress Xtra should return a S B gt 3 Standard TR F Measurement Increased levels of performance can be achieved by using time resolved fluorescence TR F TR F measurement reduces non specific background and increases probe sensitivity Optimal delay time is 30ys and gate integration time is 100us NOTE MitoXpress Xtra should return a S B gt 3 S B 10 are typical Advanced Dual Read TR F Lifetime Optimal performance can be achieved using dual read TR F in combination with subsequent ratiome
10. e 100ul to each well at an angle of 45 allowing the oil to flow the side of each well NOTE Small variations in the volume of HS Minerial Oil between 90 110yl should not adversely effect the readings using MitoXpress Xtra P17 P18 APPENDIX C TROUBLE SHOOTING Extensive literature including Protocols Application Notes Videos Publications and email technical support is also available through our website www luxcel com GENERAL NOTES AND RECOMMENDATIONS Storage and Stability On receipt the MitoXpress Xtra reagent should be stored between 2 to 8 C see Use Before date on vial Reconstituted probe stock can be stored in the dark between 2 to 8 C for several days or stored as aliquots in water at 20 C for use within one month avoid freeze thaw Plate Reader A fluorescence plate reader capable of measuring excitation at 380nm and emission at 650nm and having plate temperature control is required Plates We recommend 96 or 384 well black wall clear bottom TC plates although standard clear wall PS plates for cell culture may also be used Temperature We recommend the use of a plate block heater for plate preparation to maintain a temperature of 37 C Pre warm the fluorescence plate reader to measurement temperature and ensure that all culture media and stock solutions to be used in the assay are pre warmed at 37 C prior to use Signal Optimisation and Use of Controls We recommend performing a signal optimisati
11. gents to measure glycolysis LDH JC 1 MMP V ROS and cellular ATP For example MitoXpress Xtra in combination with Luxcel s pH Xtra Glycolysis Assay Cat No PH 100 allows the simultaneous real time measurement of mitochondrial respiration and glycolysis and analysis of the metabolic phenotype of cells and the shift flux between the two pathways under pathological states Add Oil Figure 1 Flow diagram showing preparation and use of MitoXpress Xtra Oxygen Consumption Assay HS Method P3 P6 PLATE READER SET UP MEASUREMENT PARAMETERS MitoXpress Xtra reagent is a chemically stable and inert biopolymer based cell impermeable oxygen sensing fluorophore Deoxygenated 0 6 Fold Increase 0 4 i Air saturated 0 2 gt yan o c O D o9 T w E x o Z 0 T T T 300 350 400 450 500 550 600 625 650 675 Wavelength nm Wavelength nm Figure 2 Excitation and Emission spectra of MitoXpress Xtra Left panel shows normalised excitation Ex 360 400nm Peak 380nm Right panel shows emission Em 630 680nm Peak 650nm in oxygenated and deoxygenated conditions INSTRUMENTS AND SETTINGS Three fluorescence modalities can be successfully used with the MitoXpress Xtra Oxygen Consumption Assay HS Method depending on plate reader type and instrument setup as follows 1 Basic Intensity measurement 2 Standard Time resolved fluorescence measurement TR F and 3 Advanced Dual read R
12. on Gemini Monochromator based Top or bottom read n a n a Intensity Prompt Ex 380nm Em 650nm Hidek SENSE CHAMELEON Filter based Top or bottom read 30 100us n a TR F Ex 390 20nm Em 660 10nm Thermo Varioskan Fluoroscan Ascent Monochromator Filter based Top or bottom read 30 100us n a TR F pqg Notes Assay specific protocols and notes are available from manufacturer for MitoXpress Xtra Assay specific protocols in development contact TechSupport Luxcel com TR F head must be installed Ex 380nm Em 650nm APPENDIX B HS MINERIAL OIL PIPETTING TIPS HS Mineral Oil is provided in an easy to use dropper bottle for convenience although we recommend a repeater pipette for routine use Al Figure 4 Add Oil e Dropper Bottle Invert the pre warmed dropper bottle and apply gentle pressure just sufficient to prime the oil in the bottle tip Apply two 2 drops to each well touching each drop as it is formed to the side of the well to allow it to run down onto the surface of the culture media e Repeater Pipette Use of a repeater pipette saves time and helps to maintain more precise incubation times Prepare the repeater syringe tip by trimming 3 4mm off the tip at a 45 angle Remove the internal nozzle cap from the dropper bottle and slowly pick up the pre warmed HS Mineral Oil avoid pipetting up and down as this can cause bubbles and dispens
13. on check especially for first time users and inclusion of blank and optional additional control wells as described Pipetting HS Oil Take care when dispensing the HS Mineral Oil to avoid bubbles Apply HS Mineral Oil allowing it to run down the inside surface of each well Do not shake or rapidly aspirate the HS Mineral Oil General Assay Set Up Pipetting and Aspirating Prepare your assay materials and work space in advance Take care not to disrupt the cell monolayer adherent cells during pipetting and aspirating Work rapidly once the MitoXpress Xtra reagent has been added to reduce the potential for assay variability Cell Type and Cell Density Since the MitoXpress Xtra reagent measures extracellular Oxygen Consumption the amount of signal change will be directly dependent on the rate of cellular respiration of the cell type being measured We recommend using as high a cell density per well as practical as a starting point and reducing cell numbers as required Not all cell types may consume sufficient oxygen for detection SIGNAL TO BLANK S B OPTIMISATION For most fluorescence plate readers set up according to Appendix A Instrument Settings MitoXpress Xtra should return a signal to blank ratio gt 3 Higher readings are expected with TR F and dual read TR F measurement The following options may be helpful to improve S B if the determine ratio is not as high as expected Increase Gain PMT setting or flash energy Adjust
14. rs set up according to Appendix A Instrument Settings MitoXpress Xtra should return a S B 2 3 Higher readings are expected with TR F and dual read TR F measurement NOTE See also Appendix C Trouble Shooting 1 2 3 4 Media Media Media Media MitoXpress Xtra MitoXpress Xtra MitoXpress Xtra MitoXpress Xtra Oil Oil Oil Oil Media Media Media Media Oil Oil Oil Oil P7 P8 PERFORMING THE OXYGEN CONSUMPTION ASSAY CELL CULTURE AND PLATING NOTE Always leave two wells H11 and H12 free from the addition of MitoXpress Xtra reagent as Blank Controls e For Adherent cells seed cells in a 96 well plate at a density typically 40 000 80 000 cells well in 200ul culture medium Incubate overnight in a CO incubator at 37 C e For Suspension cells seed on the day of assay in 150p culture medium at a density of 4 x 10 ml Visit our website www luxcel com for more information on the use of MitoXpress Xtra with permeabilised cells 3D cultures tissues spheroids Raft and scaffolds isolated enzymes bacteria yeasts and moulds PRE ASSAY PREPARATION e Reconstitute the contents of the MitoXpress Xtra vial in 1ml of water PBS or culture media gently aspirating 3 4 times Figure 3 NOTE Reconstituted probe stock can be stored in the dark between 2 to 8 C for several days or stored as aliquots in water at 20 C for use within one month avoid freeze thaw e Prepare test compo
15. stituted MitoXpress Xtra reagent to each well e Negative Controls To 2 or 3 wells F containing cells add 1pl of 150 uM Antimycin A stock solution in DMSO 10ul reconstituted MitoXpress Xtra reagent P10 Figure 6 Reading the assay plate ANALYSIS NOTE We recommend that all first time users perform a Signal Optimisation test as described Signal and Blank Control wells may also be included ASSESSING OXYGEN CONSUMPTION Plot the Blank Control well corrected MitoXpress Xtra Intensity or Lifetime values versus Time mins Figure 7 Select the linear portion of the signal profile avoiding any initial lag or subsequent plateau and apply linear regression to determine the slope OCR and correlation coefficient for each well NOTE This approach is preferable to calculating a slope from averaged profiles a E Antimycin A 60 Time min Figure 7 Typical Lifetime profile of MitoXpress Xtra for adherent cells treated with different ETC compounds including Antimycin A recommended as a Negative Control The effect of Glucose Oxidase as a positive Signal Control is illustrated schematically NOTE If using FCCP it is strongly recommended to perform a dose titration since FCCP exhibits a bell shaped response Tabulate the slope values for each test sample calculating appropriate average and standard deviation values across replicate wells If optional Signal Control wells are included the slope obtaine
16. t of extracellular oxygen consumption rates OCR with whole cell populations both adherent and suspension cells isolated mitochondria permeabilised cells and a wide range of 3D cultures including tissues small organisms spheroids scaffolds and matrixes The assay is also suitable for measurement of isolated enzymes bacteria yeasts and moulds Scientists at Luxcel Biosciences developed the oxygen sensing fluorophore known as MitoXpress Xtra to overcome the limitations of specialised low throughput instrumentation that were historically used to measure oxygen eg Clark electrode The MitoXpress Xtra reagent is chemically stable and inert water soluble and cell impermeable making it the ideal and scalable mix and measure reagent for use in a wide range of cell culture conditions all measured using a fluorescence plate reader In this assay MitoXpress Xtra is quenched by 0 through molecular collision and thus the amount of fluorescence signal is inversely proportional to the amount of extracellular 02 in the sample Rates of oxygen consumption are calculated from the changes in fluorescence signal over time The reaction is non destructive and fully reversible neither MitoXpress Xtra nor O2 are consumed facilitating measurement of time courses and drug treatments Luxcel s flexible plate reader format allows multiparametric or multiplex combination with Luxcel s other products as well as combination with commonly available rea
17. ter assay for assessing drug induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays Hynes J et al Toxicol Jn Vitro 2012 Mar 27 2 560 569 Comparative bioenergetic assessment of transformed cells using a cell energy budget platform Zhdanov AV et al Integr Biol 2011 3 1135 1142 High throughput assay to measure oxygen consumption in digitonin permeabilized cells of patients with mitochondrial disorders Jonckheere Al et al Clin Chem 2010 56 3 424 431 Fluorescent pH and oxygen probes of the assessment of mitochondrial toxicity in isolated mitochondria and whole cells Hynes J et a Curr Protoc Toxicol 2009 May Chapter 2 Unit 2 16 Analysis of mitochondrial function using phosphorescence oxygen sensitive probes Will Y et al Nature Protocols 2007 1 6 2563 2572 Circumventing the Crabtree effect replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants Marroquin LD et al Toxicol Sci 2007 97 2 539 547 Investigation of drug induced mitochondrial toxicity using fluorescence based oxygen sensitive probes Hynes J et al Toxicol Sci 2006 92 1 186 200 RELATED PRODUCTS e pH Xtra Glycolysis Assay Cat No PH 100 e MitoXpress Intra Intracellular O2 Assay Cat No MX 300 e GreenLight 960 Microbial Detection Assay Cat No GL 960 P21 Luxcel Biosciences Limited Suite
18. tric Lifetime calculation to maximise dynamic range Figure 10 NOTE MitoXpress Xtra should return a S B 2 3 and S B up to 60 are possible Optimal dual delay and gate integration times e Integration window 1 30us delay D1 30us measurement time W1 e Integration window 2 70us delay D2 30us measurement time W2 DUAL READ TR F AND LIFETIME ILLUSTRATED Dual read TR F and subsequent Lifetime calculation allows measurement of the rate of fluorescence decay of the MitoXpress Xtra reagent and can provide measurements of oxygen consumption that are more stable and with a wider dynamic range than measuring signal Intensity NOTE S B for Integration window 2 is recommended to be gt 10 to allow accurate Lifetime calculation gt gt n n c c D D 3 gkah E Figure 10 Illustrating dual read TR F measurement Use the dual intensity readings to calculate the corresponding Lifetime us using the following transformation Lifetime us T D2 D1 In W1 W2 Where W1 and W2 represent the two dual measurement windows and D1 and D2 represent the delay time prior to measurement of W1 and W2 respectively This provides Lifetime values in microsecond units us at each measured time point for each individual sample Figure 10 NOTE Lifetime values should be in the range 22 to 68us and should only be calculated from samples containing MitoXpress Xtra reagent Lifetime values should not be calculated from blank wells
19. unds controls and dilutions as desired Typical controls are Antimycin A Complex III inhibitor FCCP ETC uncoupler and Glucose Oxidase GOx positive signal control NOTE We recommend that all culture media and stock solutions to be used in the assay are pre warmed at 37 C prior to use Use a plate block heater for plate preparation and pre warm the fluorescence plate reader to measurement temperature Figure3 Reconstitution of MitoXpresse Xtra vial TYPICAL ASSAY To assess Oxygen Consumption or to investigate the effect of a compound on electron transport chain function ETC oxidative phosphorylation cells are treated immediately prior to measurement NOTE We recommend the use of triplicate wells for each treatment STEP 1 Remove spent culture medium from all assay wells and replace with 150ul of fresh culture media Figure 4 NOTE We recommend always leaving two wells H11 and H12 free from the addition of MitoXpress Xtra reagent for use as Blank Controls Add 150ul of fresh culture media to these Blank Control wells also STEP 2 Add 10ul reconstituted MitoXpress reagent to each well except those wells for use as Blank Controls Add 10ul of fresh culture media to these Blank Control wells NOTE If plating a full 96 well plate of assays we recommend combining Step 1 and Step 2 by adding the 1ml of reconstituted MitoXpress Xtra reagent to 15ml pre warmed fresh culture media and using a multi channel pipette to add 15
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