pH-Xtra Glycolysis Assay
Contents
1. 11 ASSESSING OXYGEN CONSUMPTION ccs pire entera tnde 11 TITRATION OF CELL SEEDING DENSITY cette 12 5 13 CALIBRATION OF PH XTRA GLYCOLYSIS ASSAY TO A PH SCALE 14 APPENDIX INSTRUMENT SETTINGS se 15 APPENDIX B TROUBLE SHOOTING e 18 REFERENCE 20 RELATED PRODUCTS eed 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 pH Xtra Reagent 1 vial 4 C Respiration Buffer 1 tablet Room Temp STORAGE AND STABILITY The pH Xtra reagent should be stored as follows Dry material between 2 to 8 C see Use Before date on vial e Reconstituted pH Xtra reagent can be stored the dark between 2 to 8 C for several days reconstituted in water and stored as aliquots at 20 C for use within one month avoid freeze thaw The Respiration Buffer tablet should be stored as follows e Dry material at room temperature see Use Before date on packaging e Reconstituted and filter sterilised product can be stored between 2 to 8 C ADDITIONAL ITEMS REQUIRED e Fluorescence plate reader with suitable filter and plate temperature control e 96 well black wall clear bot2. PLATE READER SET UP MEASUREMENT PARAMETERS pH Xtra reagent is a chemically stable and inert cell impermeable H sensing fluorophore Fold Increase NJ UJ A ON N e n D ER Uo o E o 2 300 320 340 360 380 400 420 440 460 480 500 570 590 610 630 650 670 690 710 Wavelength nm Wavelength nm Figure 2 Excitation and Emision spectra of pH Xtra Left panel shows normalized excitation Ex 340 410nm Peak 360 380nm Right panel shows emission maxima Em 590 615 and 690nm fold increase between pH6 0 and pH7 5 INSTRUMENTS AND SETTINGS Two fluorescence modalities can be optimally used with the pH Xtra Glycolysis Assay depending on plate reader type and instrument setup as follows 1 Standard Time resolved fluorescence measurement TR F and 2 Advanced Dual read Ratiometric TR F measurement Lifetime calculation 1 pH Xtra Glycolysis Assay may also be used in non TR F intensity mode on some plate readers although we recommend running the Signal Optimisation protocol and optimising cell seeding density NOTE Further details including instrument filter selection and measurement settings can be found in P6 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 Respirati
3. e Prepare test compounds controls and dilutions as desired Typical controls are oxamic acid inhibitor decrease ECA FCCP ETC uncoupler increases ECA and glucose oxidase GOx signal control NOTE We recommend that all culture media and stock solutions to be used in the assay are pre warmed at 37 prior to use Use a plate block heater for plate preparation and pre warm the fluorescence plate reader to measurement temperature Figure 3 Reconstitution of pH Xtra vial TYPICAL ASSAY To assess Extracellular Acidification ECA or to investigate the effect of a treatment on glycolytic flux 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 wash cells twice 2x using 100ul of Respiration Buffer per well for each wash Figure 4 After removing the second wash replace with 150ul of fresh Respiration Buffer NOTE We recommend always leaving two wells H11 and H12 free from the addition of pH Xtra reagent for use as Blank Controls Add 150yl of Respiration Buffer to these Blank Control wells also STEP 2 Add 10yl reconstituted pH Xtra reagent to each well except those wells for use as Blank Controls Add 10ul of Respiration Buffer to these Blank Control wells NOTE If plating a full 96 well plate of assays we recommend simplifying Step 1 and 2 by preparing a stock solution containing the 1ml
4. pH Xtra Glycolysis Assay For the measurement of Extracellular Acidification ILLUMINATING DISCOVERY E L d 4 e a gt P hi E P s gt 2 d ri Ph pw idm h C E ESSN A aaa pH Xtra Glycolysis Assay For the measurement of Extracellular Acidification For use with K Adherent cells V e Suspension cells e Permeabilised cells e 3D cultures tissues spheroids e RAFT and scaffolds TABLE CONTENTS GENERAL INFORMATION c 3 MATERIALS SUPPLIED m an wang a ataga a aa da a an ed 3 STORAGE AND STABILITY nanak saga aan an gang ag awan docebo sahe nega mapa nenge nan 3 ADDITIONAL ITEMS REQUIRED iiis 3 OPTIONAL ITEMS NOT SUPPLIED 3 DESCRIPTION e agak aaa aaa aana E a a ai 4 HO CEPIT GRAN TS aa Aah 9 PLATE READER SET 6 MEASUREMENT PARAMETERS ciiam atum 6 INSTRUMENTSAND SET TAN GS 6 SIGNAL OPTIMISATION teint tette diretta tincdunt uetus 7 PERFORMING THE OXYGEN CONSUMPTION ASSAY 8 GELE CULTURE AND PLATING uocatus aque tete edendo emot uet 8 PRESASSAT PREPARATION ao ot ES 8 M EN E E E E 9 unie m
5. 30ys Dual read TR F Lifetime Dual read TR F Lifetime Ex 340 50 TR EX L Em 615 10nm BP 615 Ex 340 40nm Em 620 10nm BMG Labtech PHERAstar FS Filter based Top read HTRF Module 100 30ys 300 30ys Dual read TR F Lifetime Ex 337nm Em 620nm BMG Labtech FLUOstar Optima POLARstar Optima Filter based Top or bottom read 100 100us n a TR F Ex 340 50 TR EX L Em 615 10nm BP 615 Perkin Elmer VICTOR series X4 X5 Filter based Top read 100 30ys 300 30ys Dual read TR F Lifetime Ex 340 40nm D340 Em 615 8 5nm D642 Perkin Elmer EnVision Filter based Top read 100 50ys 300 50ys Dual read TR F Lifetime Ex 340 60nm X340 Em 615 8 5nm M615 Perkin Elmer EnSpire Monochromator Top or bottom read 100 100us n a TR F Ex 380nm Em 615nm BioTek Synergy H4 HT Neo 2 Cytation 3 Filter based Top or bottom read 100 30ys 300 30ys Dual read TR F Lifetime Ex 360 40nm Em 620 10nm BioTek MX H1m Tecan Infinite Safire Genios Pro Mol Devices SpectraMax Flexstation Gemini Notes Monochromator Top or bottom read Filter based Monochromator Top or bottom read Monochromator based Top or bottom read 100 100ys n a 100 100ys n a 50 250us n a TR F Assay specific protocols and notes are available from manufact
6. reagent and can provide measurements of extracellular acidification that are more stable and with a wider dynamic range than measuring signal Intensity or standard TR F NOTE S B for Integration window 2 is recommended to be gt 10 to allow accurate Lifetime calculation gt gt c c D D 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 W represent the two dual measurement windows and D and D represent the delay time prior to measurement of W1 and respectively This provides Lifetime values in microsecond units ps at each measured time point for each individual sample Figure 10 NOTE Lifetime values should be in the range 200 for cells assayed in respiration buffer at approx pH7 4 increasing up to 400us upon acidification and should only be calculated from samples containing pH Xtra reagent Lifetime values should not be calculated from blank wells P16 RECOMMENDED INSTRUMENT AND MEASUREMENT SETTINGS Instrument Optical Configuration Integration 1 D4 W1 Integration 2 D2 W2 Mode Ex nm Em nm BMG Labtech FLUOstar Omega POLARstar Omega BMG Labtech CLARIOstar Filter based Top or bottom read Hybrid filter based Top or bottom read 100 30ys 300 30ys 100 30ys 300
7. Glycolysis Assay depending on plate reader type and instrument setup NOTE We strongly recommend only using fluorescence plate readers equipped with temperature control Standard TR F Measurement Measurement using time resolved fluorescence TR F provides flexibility to use a wide range of commonly available plate readers TR F measurement reduces non specific background and increases probe sensitivity Optimal delay time is 100ps and gate integration time is 100 5 NOTE pH Xtra should return a S B 23 Advanced Dual Read TR F Lifetime Optimal performance can be achieved using dual read TR F in combination with subsequent ratiometric Lifetime calculation to maximise dynamic range Figure 10 and to express ECA as a function of H NOTE pH Xtra should return a S B gt 10 Optimal dual delay and gate integration times e Integration window 1 100us delay D1 30s measurement time W1 e Integration window 2 300us delay D2 30us measurement time Wa pH Xtra Glycolysis Assay may also be used in non TR F Intensity mode on some plate readers although we recommend running the Signal Optimisation protocol to confirm an acceptable S B and optimising cells seeding density Figure 7 Users may see better performance using filter based plate readers P15 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 pH Xtra
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9. of reconstituted pH Xtra reagent added to 15ml pre warmed Respiration Buffer and using a multi channel pipette to add 150 of this diluted pH Xtra stock to each well Add 150 of Respiration Buffer only no pH Xtra to each Blank Control well Figure 4 Aliquoting fresh Respiration Buffer pH Xtra 10 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 STEP 4 Read the plate immediately in a fluorescence plate reader with the set up as described in Appendix A Instrument Settings Figure 5 The plate should be measured kinetically for 120 minutes When the measurement is completed remove the plate and save measured data to file Optional Controls Figure 5 Reading the assay plate e Signal Controls Leave 2 or 3 wells free from the addition of cells for use as Signal Control wells Add 1500 of Respiration Buffer 10pl of reconstituted pH Xtra reagent to each well e Positive Signal Controls Leave 2 or 3 wells free from the addition of cells for use as Positive Control wells Add 150yl of fresh Respiration Buffer of 1 mg ml glucose oxidase stock solution in water 10ul reconstituted pH Xtra reagent to each well e Negative Controls To 2 or 3 wells containing cells washed and refreshed with 150ul of Respiration Buff
10. A This performance facilitates sensitive robust microtitre plate based measurements thereby overcoming many of the problems associated with the more cumbersome potentiometric pH approach Rates of extracellular acidification are calculated from changes in fluorescence signal over time and as the measurement is non destructive and fully reversible pH Xtra reagent is not consumed measurement of time courses and multiple drug treatments are possible Luxcel s flexible plate reader format also allows multiparametric or multiplex combinations with Luxcel s other products and with other commonly available reagents thereby facilitating parallel kinetic measurements of parameters such as ECA mitochondria membrane potential Vm O2 consumption or ROS generation followed by end point measure of parameters such as ATP content or cell membrane integrity all on the same test cells For example the combination of Luxcel s MitoXpress Xtra Oxygen Consumption Assay HS Method Cat No MX 200 and pH Xtra Glycolysis Assay allows the simultaneous real time measurement of the interplay between of mitochondrial respiration and glycolysis This facilitates the determination of a cell s metabolic phenotype and the quantification of perturbations in the balance between glycolysis and oxidative phosphorylation under various stimuli or pathological states Aliguot Figure 1 Flow diagram showing preparation and use of pH Xtra Glycolysis Assay Read
11. Y CELL CULTURE AND PLATING NOTE Always leave two wells H11 and H12 free from the addition of pH Xtra reagent as Blank Controls e For Adherent cells seed cells in a 96 well plate at a density typically 30 000 80 000 cells well 20011 culture medium NOTE For new cell types we recommend setting up a titration to select the optimum cell seeding density see Figure 7 For Suspension cells seed on the day of assay in 150pl culture medium at a density of approx 250 000 500 000 cells well Visit our website www luxcel com for more information on the use of pH Xtra with a range of cell systems PRE ASSAY PREPARATION NOTE Where cells are cultured in a CO incubator overnight it is important to purge the media and plasticware of CO prior to conducting the pH Xtra Glycolysis Assay as residual CO may contribute to acidification Perform a CO purge by incubating cells in a CO2 free incubator at 37 C with 95 humidity approx 3 hours prior to performing the Glycolysis Assay measurement e Reconstitute Respiration Buffer tablet in 50ml of water pH adjust to approx pH7 4 and filter sterilise using a 0 22um filter Reconstitute transparent contents of the pH Xtra vial in 1ml of Respiration Buffer gently aspirating 3 4 times NOTE Reconstituted pH Xtra reagent 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
12. er add of 750 mM oxamic acid stock solution in water 10pl reconstituted 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 EXTRACELLULAR ACIDIFICATION Plot the Blank Control well corrected pH Xtra Intensity or Lifetime values versus Time Figure 6 Select the linear portion of the signal profile avoiding any initial lag or subsequent plateau and apply linear regression to determine the slope ECA and correlation coefficient for each well NOTE This approach is preferable to calculating a slope from averaged profiles GOx Antimycin A FCCP Untreated N _ 7 20 D E Oxamic Acid 90 120 Time mins Figure 6 Typical Lifetime profile of pH Xtra for adherant cells treated with typical control compounds including oxamic acid 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 dose 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 obtained for the Signal Control sample without cells should be subtracted from all test
13. igure 8 l6 Oxygen Consumption ol Glycolysis n Rotenone X Antimycin FCCP Oxamate Untreated Figure 8 Cellular Energy Flux for HepG2 cells treated with a combination of drug compounds modulating the ETC or inhibiting lactate production shown as a percentage relative to untreated control cells Comparative measurements with pH Xtra glycolysis and MitoXpress Xtra oxygen consumption show the shift between glycolysis and mitochondrial respiration and the cellular control of energy ATP measured 1h post treatment using Promega Cell Titer Glo P13 CALIBRATION OF pH XTRA GLYCOLYSIS ASSAY TO A pH H SCALE It is possible to express Extracellular Acidification ECA as a function of pH H versus time This is achieved by first creating a calibration standard curve by measuring TR F intensity or preferably Lifetime values selecting stabilised readings over a 30 minute read from a range of pH buffered standards at the appropriate assay temperature see Figure 9 Select the linear portion of the standard curve and apply linear regression to determine the calibration function See Hynes et al 2009 N 5 wo E Figure 9 pH XTRA reagent calibration in Lifetime scale at 30 and 37 using pH buffered PBS at increments of 0 2 across a pH range 6 0 7 5 P14 APPENDIX A INSTRUMENT SETTINGS Two fluorescence modalities can be optimally used with the pH Xtra
14. n between 360nm and 390nm see Figure 2 and emission at 615nm and having plate temperature control is required We strongly recommend using TR F measurement Temperature We recommend the use of a plate block heater for plate preparation to maintain a temperature of 37 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 prior to use Signal Optimisation and Use of Controls We recommend performing a signal optimisation check especially for first time users and inclusion of blank and optional additional control wells as described 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 pH Xtra reagent has been added to reduce the potential for assay variability Re check pH of Respiration Buffer prior to use Cell Type and Cell Density Since the pH Xtra reagent measures Extracellular Acidification the amount of signal change will be directly dependent on the rate of glycolytic flux of the cell type being measured We recommend using a medium to high cell density per well as a starting point and reducing cell numbers as P18 required See Figure 7 SIGNAL BLANK S B OPTIMISATION For most fluorescence plate readers set up according to Appendi
15. on Buffer tablet in 50ml of water warm to assay temperature 37 C pH adjust to approx pH7 4 and filter sterilise using a 0 22um filter Reconstitute transparent contents of the pH Xtra vial in 1ml of Respiration Buffer gently aspirating 3 4 times NOTE Reconstituted pH Xtra reagent 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 1501 pre warmed Respiration Buffer to each well A1 A4 B1 B4 STEP 3 Add reconstituted pH Xtra reagent to 4 of the replicate wells A1 A4 and 10yl Respiration Buffer to the remaining replicate wells B1 B4 STEP 4 Read plate immediately in a fluorescence plate reader over 30 minutes read every 2 3 minutes STEP 5 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 TR F plate readers set up according to Appendix A Instrument Settings pH Xtra should return a S B 23 NOTE See also Appendix B Trouble Shooting Respiration Buffer Respiration Buffer Respiration Buffer Respiration Buffer pH Xtra pH Xtra pH Xtra pH Xtra Respiration Buffer Respiration Buffer Respiration Buffer Respiration Buffer P7 8 PERFORMING THE GLYCOLYSIS ASSA
16. tom TC plates or standard PS plates for cell culture OPTIONAL ITEMS NOT SUPPLIED e Plate block heater for plate preparation e 0 22um sterilization filter pH meter and acid base for adjustment SUPPORT e Visit our website www luxcel com 1 May also be used in a 384 well format with one vial of reagent sufficient for 200 wells 2 1mM K phosphate 20mM Glucose 70mM NaCl 50mM KCI 0 8mM MgSOs4 2 4mM P3 DESCRIPTION The pH Xtra Glycolysis Assay from Luxcel Biosciences is an easy to use highly flexible 96 or 384 well fluorescence based approach for the direct real time kinetic analysis of extracellular acidification rates ECA ECAR As lactate production is the main contributor to this acidification ECA measurements are a convenient and informative measure of cellular glycolytic flux Such measurements offer an important insight into the central role played by altered glycolytic activity in a wide array of physiological and pathophysiological processes including cellular adaptation to hypoxia and ischemia and the development and progression of tumorigensis The pH Xtra reagent is chemically stable and inert water soluble and cell impermeable It exhibits a positive signal response increased signal with increased acidification across the biological range pH6 7 5 which coupled with its spectral and response characteristics make pH Xtra the ideal choice for flexible high throughput assessment of EC
17. urer for pH Xtra Assay specific protocols in development contact TechSupport Luxcel com Where TR F indicated a TR F module must be installed Ex 380nm Em 615nm Ex 380 20nm Em 615 10nm Ex 380nm Em 615nm Note Choose filter based optical configuration where available Instruments with recommended Dual read TR F measurement mode can alternatively be set up using Standard TR F measurement mode if desired APPENDIX B TROUBLE SHOOTING Extensive literature including 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 pH Xtra reagent should be stored between 2 to 8 C see Use Before date on vial Reconstituted pH Xtra reagent 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 Note pH Xtra reagent diluted in Respiration Buffer media should be used on the same day Respiration Buffer Kit contains a single Respiration Buffer tablet sufficient for 50ml 1x stock containing 1mM K phosphate 20mM Glucose 70mM NaCl 50mM KCI 0 8mM MgSOa 2 4mM CaCb Alternative media and supplements may be used as required such as unbuffered DMEM so long as care is taken to ensure a minimal buffering capacity Plate Reader A fluorescence plate reader capable of measuring excitatio
18. values P11 Data analysis templates are available from some plate reader manufacturers specifically configured to automate the analysis of Luxcel s pH Xtra Glycolysis Assay Microsoft Excel templates are also available through our website www luxcel com TITRATION OF CELL SEEDING DENSITY To determine an optimal cell seeding density for performing the pH Xtra Glycolysis Assay for new cell types seed replicate wells with a range of seeding densities typically O 10 000 20 000 40 000 60 000 and 80 000 cells well Plot the data generated as a function of intensity or Lifetime values versus time as illustrated Figure 7 N _ D prar D 20 000 10 000 90 120 Time mins Figure 7 Extracellular Acidification rate profiles ECA are shown for A549 cells seeded at 0 10 000 20 000 40 000 60 000 and 80 000 cells well In this experimental example a seeding density of 40 000 cells well was chosen for study as this provided a suitable balance between ECA response and cell availability P12 CELLULAR ENERGY FLUK ANALYSIS Multiparametric or multiplex combination of pH Xtra Glycolysis Assay together with Luxcel s MitoXpress Xtra Oxygen Consumption Assay HS Method Cat No MX 200 allows the simultaneous real time measurement of glycolysis and mitochondrial respiration and analysis of the metabolic phenotype of cells and the shift flux between the two pathways under pathological states F
19. void outer wells If tested and not resolved contact TechSupport luxcel com 19 P20 REFERENCES A high throughput dual parameter assay for assessing drug induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays Hynes J et al Toxicol In 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 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 In vitro analysis of cell metabolism using a long decay pH sensitive lanthanide probe and extracellular acidification assay Hynes J et al Analytical Biochemistry 2009 390 21 28 RELATED PRODUCTS e MitoXpress Xtra Oxygen Consumption Assay HS Method Cat No MX 200 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 2 04 Western Gateway Building Western Road Cork Ireland t 353 0 21 420 5348 e Sales luxcel com w www luxcel com i iis Ws e stad F 4 a 4 c Pm o P i gt 2 m 5 cH b aab P
20. x A Instrument Settings pH Xtra should return a S B ratio 23 The following options may be helpful to improve S B if the ratio is not as high as expected Increase Gain PMT setting or flash energy Adjust TR F focal height Increase length of integration time the same for both delay windows Repeat as top or bottom read respectively Increase volume of pH Xtra 151 Contact Instrument Supplier for further options ON FREQUENTLY ASKED QUESTIONS What do do if cannot detect any signal in wells containing cells and pH 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 Check pH of pre warmed Respiration Buffer and correct as necessary as pH can drift over time If tested and not resolved contact TechSupport luxcel com What do do if can detect a signal in wells containing cells and pH 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 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 and a
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