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 3 Last Updated 12 January 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 4 6. MATERIALS REQUIRED, NOT SUPPLIED 4 7. LIMITATIONS 5 8. TECHNICAL HINTS 5 9. MEASUREMENT PARAMETERS DUAL-READ TR-F AND LIFETIME ILLUSTRATED 7 ASSAY PREPARATION 11. CELL CULTURE AND PLATING REAGENT PREPARATIONS 9 ASSAY PROCEDURE 13. SIGNAL OPTIMISATION ASSAY PROCEDURE 11 DATA ANALYSIS 15. CALCULATIONS INSTRUMENT AND MEASUREMENT SETTINGS 14 RESOURCES 17. ADDITIONAL INFORMATION NOTES 17 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 Abcam s other products. For example, this product in combination with the Extracellular O 2 Consumption Assay (ab197243) or the Extracellular O 2 probe (ab197242) allows the simultaneous realtime 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 vial containing Glycolysis Assay reagent at 4 C in dark. Store Respiration Buffer tablet at room temperature in dry/dark (desiccant recommended). Reconstituted product can be aliquoted and stored at -20 C. Use within one month (avoid freeze thaw). 5. MATERIALS SUPPLIED Components supplied Quantity Storage Lyophilized Glycolysis Assay Reagent 1 vial +2-8ºC Respiration Buffer 1 tablet RT 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: 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 and GOx Fluorescence plate reader Discover more at 4

6 GENERAL INFORMATION 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures 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. 8. TECHNICAL HINTS 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. Complete removal of all solutions and buffers during wash steps.. Refer to the guide in the Resources section, for recommended settings for your plate reader. Perform a Signal Optimization step (especially first time users). 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. Discover more at 5

7 GENERAL INFORMATION 9. MEASUREMENT PARAMETERS The ph-sensitive reagent in Abcam s Glycolysis Assay (Cat No: ab ) 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: 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 6

8 GENERAL INFORMATION 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 Optimisation 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 Optimisation protocol and optimising cell seeding density. 10. 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. Discover more at 7

9 GENERAL INFORMATION Figure 3: Illustrating dual read TR-F measurement 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 8

10 ASSAY PREPARATION 11. CELL CULTURE AND PLATING 1. For Adherent cells: Seed cells in a 96-well plate at a density (typically 40,000 80,000 cells/well) in 200 μl culture medium. Incubate overnight in a CO 2 incubator at 37 C For Suspension cells: Seed on the day of assay in 150 μl culture medium at a density of 250, ,000 cells/well. Note: Always leave two wells (H11 and H12) free from the addition of Glycolysis Assay reagent, as Blank Controls. 12. REAGENT PREPARATIONS Reconstitute Respiration Buffer tablet in 50 ml of water, warm to assay temperature (37 C), ph adjust to approx. ph7.4 and filter sterilize using a 0.22 μm filter. Reconstitute vial containing Glycolysis Assay reagent in 1 ml of Respiration Buffer, gently aspirating 3-4 times. Prepare test compounds, controls and dilutions as desired. Typical controls are Oxamic acid (inhibitor; 750 M stock in water), FCCP (ETC uncoupler, titration recommended to establish best concentration) and Glucose Oxidase (GOx, positive signal control, 1 mg/ml in water). Discover more at 9

11 ASSAY PROCEDURE 13. SIGNAL OPTIMISATION Note: Use a plate block heater for plate preparation and pre-warm plate reader to measurement temperature (typically 37 C). 13 Reconstitute Respiration Buffer tablet in 50mL of water, warm to assay temperature (37 C), ph adjust to approx. ph7.4 and filter sterilize using a 0.22μm filter. Reconstitute vial containing Glycolysis Assay reagent in 1 ml of Respiration Buffer, gently aspirating 3-4 times 13.2 Prepare 8 replicate wells of a 96-well plate, by adding 150 µl pre-warmed culture medium to each well (A1-A4, B1-B4) Add 10 µl reconstituted Extracellular O 2 Consumption Reagent to 4 of the replicate wells (A1-A4) and 10 µl water, PBS or media to the remaining replicates wells (B1-B4) Promptly add two drops (or 100 µl) pre-warmed High sensitivity mineral oil to all eight replicate wells, taking care to avoid air bubbles 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 Media + O 2 Reagent + Oil Media + O 2 Reagent + Oil Media + O 2 Reagent + Oil Media + O 2 Reagent+ Oil B Media + Oil Media + Oil Media + Oil Media + Oil Discover more at 10

12 ASSAY PROCEDURE 14. ASSAY PROCEDURE 14 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 l 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 Test compound stock or vehicle (typically 1-10 µl) may be added at this point if desired. Note: We recommend keeping the volume of added compound low to minimize any potential effects of solvent vehicle 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 Instrument and Measurement section for instrument-specific settings and filters). Note: Sufficient cell numbers are required to produce measurable signal changes. We recommend setting up a titration to select the optimum cell seeding density. Discover more at 11

13 DATA ANALYSIS 15. CALCULATIONS 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. Discover more at 12

14 DATA ANALYSIS Figure 4: Typical Lifetime profile of Glycolysis Assay for adherent 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. Discover more at 13

15 RESOURCES 16. INSTRUMENT AND MEASUREMENT SETTINGS Instrument Optical Configuration Integration 1 (D1 / W1) Integration 2 (D2 / W2) BMG Labtech:* FLUOstar Omega POLARstar Omega BMG Labtech:* CLARIOstar** BMG Labtech:* PHERAstar FS BMG Labtech:* FLUOstar Optima / POLARstar Optima Perkin Elmer: VICTOR series / X4, X5 Perkin Elmer: EnVision Perkin Elmer: EnSpire BioTek:* Synergy H1, H4, HT, Neo, 2 Cytation 3** BioTek: MX, H1m Tecan: Infinite / Safire / Genios Pro Mol. Devices: SpectraMax / Flexstation / Gemini Filter-based Top or bottom read Hybrid - filter based Top or bottom read Filter-based Top read (HTRF Module) Filter-based Top or bottom read Filter-based Top read Filterbased Top read Monochromator Top or bottom read Filter-based Top or bottom read Monochromator Top or bottom read Filterbased/Monochromator Top or bottom read Monochromator-based Top or bottom read 100 / 30μs 300 / 30μs 100 / 30μs 300 / 30μs 100 / 30μs 300 / 30μs 100 /100μs n/a 100 / 30μs 300 / 30μs 100 / 50μs 300 / 50μs 100 /100μs n/a 100 /30μs 300 / 30μs 100 /100μs n/a 100 /100μs n/a 50 / 250μs n/a Mode*** Dual-read TR-F (Lifetime) Dual-read TR- F (Lifetime) Ex (nm) Em (nm) Ex 340 ± 50nm (TR-EX L) Em 615 ± 10nm (BP-615) Ex 340 ± 40nm Em 620 ± 10nm Dual-read TR- Ex 337nm F Em 620nm (Lifetime) TR-F Ex 340 ± 50nm (TR-EX L) Em 615 ± 10nm (BP-615) Dual read TR- F (Lifetime) Dual-read TR-F (Lifetime) TR-F Dual read TR- F (Lifetime) TR-F TR-F TR-F Ex 340 ± 40nm (D340) Em 615 ±8.5nm (D642) Ex 340 ± 60nm (X340) Em 615 ±8.5nm (M615) Ex 380nm Em 615nm Ex 360 ±40nm Em 620 ± 10nm Ex 380nm Em 615nm Ex 380 ± 20nm Em 615 ± 10nm Ex 380nm Em 615nm Discover more at 14

16 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 15

17 RESOURCES Cellular Energy Flux Analysis Multiparametric (or multiplex) combination of Extracellular O 2 Consumption Assay together with Abcam s Glycolysis Assay (Cat No: ab197244) 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). 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 and Glycolysis Assay, show the shift between mitochondrial respiration and glycolysis and the cellular control of energy (ATP). Discover more at 16

18 RESOURCES 18. NOTES Discover more at 17

19 RESOURCES Discover more at 18

20 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 19