ab Glycolysis Assay [Extracellular Acidification]

Transcription

1 ab Glycolysis Assay [Extracellular Acidification] Instructions for Use For the measurement of extracellular acidification by cell populations, 3D culture models and small organisms This product is for research use only and is not intended for diagnostic use. Version 6 Last Updated 12 May 2015

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 5 6. MATERIALS REQUIRED, NOT SUPPLIED 5 7. LIMITATIONS 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION CELL CULTURE AND PLATING 9 ASSAY PROCEDURE and DETECTION 11. MEASUREMENT PARAMETERS DUAL-READ TR-F AND LIFETIME ILLUSTRATED SIGNAL OPTIMISATION ASSAY PROCEDURE 15 DATA ANALYSIS 15. CALCULATIONS 16 RESOURCES 16. INSTRUMENT AND MEASUREMENT SETTINGS ADDITIONAL INFORMATION GENERAL RECOMMENDATIONS FAQs 23 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Glycolysis Assay [Extracellular Acidification] (ab197244) is an easy mix-and-measure, 96 or 384 well fluorescence plate reader-based approach for the analysis of extracellular acidification (ECA/ECAR). As lactate production is the main contributor to this acidification, Glycolysis Assay (ab197244) is a convenient and informative measuring tool 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 tumorigenesis. The ph-sensitive 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 (ph 6 7.5). This performance, coupled with its spectral characteristics, make this kit the ideal choice for flexible, high-throughput assessment of extracellular acidification, 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 fully reversible, measurement of time courses and multiple drug treatments are possible. The flexible plate reader format, allows multiparametric or multiplex combination with other products within this range. For example, this product in combination with the Extracellular O 2 Consumption Assay (ab197243) or the Extracellular O 2 probe (ab197242) 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. Discover more at 2

4 INTRODUCTION 2. ASSAY SUMMARY Figure 1: Flow diagram showing preparation and use of Glycolysis Assay [Extracellular Acidification] (ab197244). Discover more at 3

5 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at 4ºC in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 5. Aliquot components in working volumes before storing at the recommended temperature. Reconstituted components are stable for 1 months. Discover more at 4

6 GENERAL INFORMATION 5. MATERIALS SUPPLIED Item Lyophilized Glycolysis Assay Reagent Amount Storage condition (Before Preparation ) Storage condition (After Preparation ) 1 vial +2-8ºC -20 C Respiration Buffer 1 tablet RT* -20 C *Store Respiration Buffer table at RT in dry/dark (desiccant recommended). 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: MilliQ water or other type of double distilled water (ddh 2 O) Sterilizing filter 0.22 µm. Plate heater CO 2 -free Incubator 96-well (black wall) clear bottom TC+ plates or standard clear polystyrene plates for cell culture. Cells and cell culture medium Compounds of interest, such as Oxamic acid, FCCP (ab120081) and glucose oxidase (GOx) Fluorescence microplate reader see section 16 for recommended filters and settings Discover more at 5

7 GENERAL INFORMATION 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not use kit or components if it has exceeded the expiration date on the kit labels. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. Discover more at 6

8 GENERAL INFORMATION 8. TECHNICAL HINTS This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Keep enzymes, heat labile components and samples on ice during the assay. Make sure all buffers and solutions are at room temperature before starting the experiment. Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Make sure you have the right type of plate for your detection method of choice. Make sure the heat block/water bath and microplate reader are switched on. Refer to the guide in the Resources section, for recommended settings for your plate reader. Perform a Signal Optimization step (especially first time users). Discover more at 7

9 ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening. 9.1 Respiration Buffer: Reconstitute Respiration Buffer table in 50 ml of ddh 2 O. Warm to assay temperature (37 C), adjust to ph = 7.4 and filter sterilize using a 0.22 µm filter. Aliquot buffer so that you have enough volume to perform the desired number of assays. Store at - 20 C protected from light. 9.2 Glycolysis Assay Reagent: Reconstitute in 1 ml of Respiration Buffer by gently aspirating 3 4 times. Aliquot reagent so that you have enough volume to perform the desired number of assays. Store at - 20 C protected from light. Once the probe is thawed, use with one month. Discover more at 8

10 ASSAY PREPARATION 10. CELL CULTURE AND PLATING This assay is for live cells only. Sufficient cell numbers are required to produce measurable signal changes. For new cell types, we recommend setting up a titration to select the optimum cell seeding density Adherent cells: Seed cells in a 96-well plate in 200 µl of culture medium at a density of 30,000 80,000 cells/well Incubate overnight in a CO2 incubator at 37 C Suspension cells: Seed cells on the day of assay in 150 µl of culture medium at a density of 250, ,000 cells/well. Discover more at 9

11 ASSAY PROCEDURE and DETECTION 11. MEASUREMENT PARAMETERS The ph-sensitive reagent in this assay is chemically stable and inert, water-soluble and cell impermeable. Figure 2: Excitation and Emission spectra of Glycolysis Assay. Left panel shows normalized excitation (Ex = nm; Peak nm). Right panel shows emission maxima (Em = 590, 615 and 690nm) fold increase between ph6.0 and ph7.5. Two fluorescence modalities can be successfully used with the Glycolysis Assay, depending on plate reader type and instrument setup, as follows: 11.1 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 ~100 μs and gate (integration) time is 100 μs. NOTE: Signal Optimization test should return a S:B 3. Discover more at 10

12 ASSAY PROCEDURE and DETECTION 11.2 Advanced: Dual-Read TR-F (Lifetime) Optimal performance can be achieved using dual-read TR-F in combination with subsequent ratiometric lifetime calculation, to maximize dynamic range. NOTE: Signal Optimization test may return a S:B 10. Optimal dual-delay and gate (integration) times: Integration window 1:100 µs delay (D1), 30 µs measurement time (W1) Integration window 2:300 µs delay (D2), 30 µs measurement time (W2) NOTE: Glycolysis Assay may also be used in non - TR-F intensity mode on some plate readers, although we recommend running the Signal Optimization protocol and optimizing cell seeding density. Discover more at 11

13 ASSAY PROCEDURE and DETECTION 12. 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 Glycolysis Assay 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 10 to allow accurate Lifetime calculation. Figure 3: Illustrating dual read TR-F measurement Discover more at 12

14 ASSAY PROCEDURE and DETECTION Use the dual intensity readings to calculate the corresponding Lifetime (μs) using the following transformation: Lifetime (μs)[τ] =(D2-D1)/ln(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 (µs) at each measured time point for each individual sample. Note: Lifetime values should be in the range ~200µs for cells assayed in respiration buffer at approximately ph 7.4, increasing up to >400μs upon acidification, and should only be calculated from samples containing Glycolysis Assay reagent. Lifetime values should not be calculated from blank wells. Discover more at 13

15 ASSAY PROCEDURE and DETECTION 13. SIGNAL OPTIMISATION Perform this signal optimization procedure when performing the assay for first time. NOTE: Use a plate block heater for plate preparation and pre-warm plate reader to measurement temperature (typically 37 C) Prepare 8 replicate wells of a 96-well plate, by adding 150 µl pre-warmed culture medium to each well (A1-A4, B1-B4) Blank controls: always leave two wells (H11 and H12) free from the addition of Glycolysis Assay reagent Add 10 µl reconstituted Glycolysis Assay Reagent to 4 of the replicate wells (A1-A4) and 10 µl ddh 2 O, PBS or media to the remaining replicates wells (B1-B4) Read plate immediately in a fluorescence plate reader over 30 minutes (read every 2-3 minutes) Examine Signal Control well (A1-A4) and Blank Control well (B1-B4) readings (linear phase) and calculate Signal to Blank (S:B) ratio. Note: For dual read TR-F, calculate S:B for each measurement window A B Respiration buffer + Glycolysis reagent Respiration Buffer Respiration buffer + Glycolysis reagent Respiration Buffer Respiration buffer + Glycolysis reagent Respiration Buffer Respiration buffer + Glycolysis reagent Respiration Buffer Discover more at 14

16 ASSAY PROCEDURE and DETECTION 14. ASSAY PROCEDURE 14.1 PRE-ASSAY PREPARATION: Where cells are cultured in a CO 2 incubator overnight, it is important to purge the media and plasticware of CO 2 prior to conducting the Glycolysis Assay as residual CO 2 may contribute to acidification. Perform a CO 2 purge, by incubating in a CO 2 -free incubator at 37 C with 95% humidity, approx. 3 hours prior to performing the Glycolysis Assay measurement.remove spent culture medium from all assay wells and wash twice (2x), using 100 µl of Respiration Buffer per well for each wash. After removing the second wash, replace with 150 µl of Respiration Buffer Add 10 µl reconstituted Glycolysis Assay reagent to each well, except those wells for use as Blank Controls. Add 10 µl of Respiration Buffer to these Blank Control wells (Optional) Add 1 10 µl of test compound stock or vehicle if using. We recommend keeping the volume of added compound low to minimize any potential effects of solvent vehicle.typical controls are: Oxamic acid (inhibitor) = 750 µm stock in ddh 2 O FCCP (ETC uncoupler, increases ECA) = titration recommended to establish best concentration as FCCP exhibits a bell-shaped dose response. Glucose Oxidase (positive control) = 1 mg/ml in ddh 2 O 14.5 Insert the prepared plate into a fluorescence plate reader pre-set to measurement temperature (typically 37 C) Measure Glycolysis Assay signal at 1.5 min intervals for > 120 minutes using excitation and emission wavelengths of 380 nm and 615 nm respectively (see section 16 for instrumentspecific settings and filters). Discover more at 15

17 DATA ANALYSIS 15. CALCULATIONS 15.1 Subtract the values of Blank Control well from the measurement Plot the blank control well-corrected Glycolysis Assay Intensity or Lifetime values versus Time (mins) 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 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 values. Figure 4: Typical Lifetime profile of Glycolysis Assay for adherent cells, treated with typical control compounds, including Oxamic acid (negative control). The effect of GOx as a positive signal control is illustrated schematically. Discover more at 16

18 RESOURCES 16. INSTRUMENT AND MEASUREMENT SETTINGS Instrument BioTek: Cytation 3 / 5 BioTek: Synergy H1 / Neo / 2 BMG Labtech: CLARIOstar BMG Labtech: FLUOstar Omega / POLARstar Omega Perkin Elmer: VICTOR series / X4 / X5 Tecan: Infinite M1000Pro / F200Pro BioTek: Synergy HTx / Mx BMG Labtech: PHERAstar FS BMG Labtech: FLUOstar Optima / POLARstar Optima Perkin Elmer: EnVision Perkin Elmer: EnSpire Optical Configuration Filter-based Top or bottom read Filter-based Top or bottom read Filter-based Bottom read Filter-based Top or bottom read Filter-based Top read Monochroma tor / Filter-based Top or bottom read Monochroma tor / Filter-based Top or bottom read Filter-based Top or bottom read Filter-based Top or bottom read Filter-based Top read Monochroma tor based Integration 1 (D 1 /W 1 ) lntegration 2 (D 2 /W 2 ) 30 / 30μs 70 / 30μs 30 / 30μs 70 / 30μs 30 / 30μs 70 / 30μs 30 / 30μs 70 / 30μs 30 / 30μs 70 / 30μs 30 / 30μs 70 / 30μs 30 / 100μs n/a 40 / 100μs n/a 30 / 100μs n/a 40 / 100μs n/a 40 / 100μs n/a Optimum Mode Dual-read TR-F (Lifetime) Dual-read TR-F (Lifetime) Dual-read TR-F (Lifetime) Dual-read TR-F (Lifetime) Dual-read TR-F (Lifetime) Dual-read TR-F (Lifetime) TR-F TR-F TR-F TR-F TR-F Ex (nm) Em (nm) Ex 380 ± 20nm Em 645 ± 15nm Ex 380 ± 20nm Em 645 ± 15nm Ex 340 ± 50nm (TR-EX) Em 665 ± 50nm or Em 645± 10nm With LP-TR Dichroic Ex 340 ± 50nm (TR- EXL) Em 655 ± 25nm (BP- 655) Ex 340 ± 40nm (D340) Em 642 ± 10nm (D642) Ex 380 ± 20nm Em 650 ±20nm or Em 670±40nm Ex 380±20nm Em 650±15nm Ex 337 nm (HTRF Module) Em 665 nm (HTRF Module) Ex 340 ± 50nm (TR- EXL) Em 655 ± 50nm (BP- 655) Ex 340 nm±60nm (X340) Em 650 nm± 8nm (M650) Ex 380 nm±20nm Em 650 nm±20nm Discover more at 17

19 RESOURCES Tecan: Infinite M200Pro / Saffire / Genios Pro Mol. Devices: SpectraMax / Flexstation / Gemini Top read Monochroma tor / Filter-based Top or bottom read Monochroma tor based Top or bottom read 30 / 100μs n/a n/a n/a TR-F Intensity (Prompt) Ex 380±20nm Em 650±20nm Ex 380nm Em 650nm Discover more at 18

20 RESOURCES 17. ADDITIONAL INFORMATION Titrating Seeding Density Figure 5: 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. Discover more at 19

21 RESOURCES Cellular Energy Flux Analysis Multiparametric (or multiplex) combination Glycolysis Assay (ab197244) together with Extracellular O 2 Consumption Assay (ab197243) 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 6: 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 Extracellular O 2 Consumption Assay (ab197243) and Glycolysis Assay (ab197244), show the shift between mitochondrial respiration and glycolysis and the cellular control of energy (ATP). Discover more at 20

22 RESOURCES Figure 7: Cellular Energy Flux for HepG2 cells (seeded at 65,000 per well), treated with a combination of drug compounds modulating the ETC (Antimycin A [1 µm] and FCCP [2.5 µm]), shown as a percentage relative to untreated control cells. Comparative measurements with Extracellular Oxygen Consumption Assay (ab197243) and Glycolysis Assay [Extracellular acidification] (ab197244), show the shift between mitochondrial respiration and glycolysis and the cellular control of energy (ATP; measured 1h post-treatment using Abcam Luminescent ATP Detection Assay kit, (ab113849). Discover more at 21

23 RESOURCES 18. GENERAL RECOMMENDATIONS RESPIRATION BUFFER: Kit contains a single Respiration Buffer tablet sufficient for 50ml 1x stock, containing 1mM K-phosphate, 20mM Glucose, 70mM NaCl, 50mM KCl, 0.8mM MgSO4, 2.4mM CaCl2. 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 excitation 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. SIGNAL TO BLANK (S:B) OPTIMISATION: For most fluorescence plate readers, set up according to Section 16. Glycolysis Reagent should return a S:B ratio 3. The following options may be helpful to improve S:B if the ratio is not as high as expected: 1. Increase Gain (PMT) setting or flash energy 2. Adjust TR-F focal height 3. Increase length of integration time, the same for both delay windows. 4. Repeat as top or bottom-read, respectively. 5. Increase volume of Glycolysis Reagent (15µl) 6. Contact Instrument Supplier for further options. Discover more at 22

24 RESOURCES 19. FAQs What do I do if I cannot detect any signal in wells containing cells and Glycolysis Reagent (or I can detect a signal but the slope rate appears very low)? Check correct Instrument Settings (Section 16). Perform Signal Optimization. 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. What do I do if I can detect a signal in wells containing cells and Glycolysis Reagent, but the slope (rate) falls initially or is variable from well to well? 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. Discover more at 23

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28 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2015 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. RESOURCES 27