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ab140097, ab140098 & ab140099 – Extracellular O2 Probe Instructions for Use For the measurement of extracellular oxygen consumption by isolated mitochondria, cell populations, small organisms, tissues and enzymes. This product is for research use only and is not intended for diagnostic use. 1 Table of Contents 1. Introduction 3 2. Product Overview 4 3. Protocol for Cell-Based Screening of Mitochondrial Analysis 5 4. Protocol for Plate Based Analysis of Isolated Mitochondria 11 5. Assay Throughput and Performance 13 6. Assessment of Classical Mitochondrial Effectors 15 7. Compound Screening 17 2 1. Introduction Mitochondrial dysfunction is implicated in numerous disease states and is also a major mechanism of drug-induced toxicity. Oxygen consumption is one of the most informative and direct measures of mitochondrial function. Traditional methods of measuring oxygen consumption are hampered by the limitations of low throughput and high complexity. The Extracellular O2 probe assay from Abcam solves these limitations by allowing convenient, plate-based analysis of mitochondrial function. The assay is based on the ability of O₂ to quench the excited state of the Extracellular O2 probe. As the test material respires (e.g. isolated mitochondria, cell populations, small organisms, tissues and enzymes), O₂ is depleted in the surrounding solution/environment, which is seen as an increase in probe phosphorescence signal. Changes in oxygen consumption reflecting changes in mitochondrial activity are seen as changes in Extracellular O2 probe signal over time. The assay is non-chemical and reversible; a decrease in oxygen consumption (an increase in O₂ levels) is seen as a decrease in probe signal. Extracellular O2 probe is analysed on standard fluorescence plate readers using standard 96-well microtitre plates. The Extracellular O2 probe assay combines the high data quality and information content of the oxygen electrode approach, with the throughput and convenience of microtitre plate based assays. These 3 capabilities make Extracellular O2 probe the ideal tool for rapid compound screening, IC50 generation and the application of structure-activity relationship approaches. 2. Product Overview The Extracellular O2 probe is supplied as a dry, stable and soluble reagent that is easily reconstituted in ultra-pure water for simple dispensing with a pipette. One vial of Extracellular O2 is used probe per 96-well plate. The Extracellular O2 probe for plate-based extracellular analysis is provided in three different formats: 1. Extracellular O2 Probe (ab140097): comprises 1 vial of Extracellular O2 probe only. 2. Extracellular O2 Probe (with mineral oil) (ab140098): contains 1 vial of Extracellular O2 probe and 1 bottle of mineral oil. 3. Extracellular O2 Probe (with high-sensitivity oil) (ab140099): contains 1 vial of Extracellular O2 probe and 1 bottle of high-sensitivity oil. Use with ab140100 Cellular Acid Extrusion Probe to simultaneously measure oxygen consumption and pH changes on a plate-reader. For the intracellular analysis of molecular oxygen, use ab140101 Intracellular O2 Probe. 4 3. Protocol for Cell-Based Screening of Mitochondrial Analysis Assay Summary Add Cells, Extracellular O2 Probe and Compound to 96-well plate Add mineral oil to wells Read plate using fluorescence plate reader 3.1. Plate Preparation 3.1.1. Equilibrate plate temperature on a plate heater to 30°C. 3.1.2. Add 150 µL of cells to test wells (suspension/ trypsinised adherent cells). Alternatively, adherent cells may be pre-plated and 150 µL of fresh media added prior to measurement. 3.1.3. Reconstitute Extracellular O2 Probe in 1 mL ultra-pure H2O to give a 1 µM stock solution and dispense 10 µL of this stock solution into to each well. 3.1.4. For compound testing, add 1 µL of compound in an appropriate solvent to test wells. If required, incubate cells with compounds. 5 3.1.5. Add a 100 µL layer of mineral oil (preheated to 30°C) volume to each well. This increases assay sensitivity by minimizing interference from ambient oxygen. 3.2. Measurement 3.2.1. Insert the prepared plate into a fluorescence plate reader pre-set to 30°C. 3.2.2. Measure Extracellular O2 Probe signal at 1.5 min intervals for 30-200 min using excitation and emission wavelengths of 380 nm and 650 nm respectively. (Readers time-resolved mode, equipped may achieve with a improved performance using delay and gate time of 30 and 100 µsec). 3.2.3. Cell respiration causes a reduction in the concentration of dissolved oxygen in the sample, resulting in an increase in Extracellular O2 Probe signal. Note: Sufficient cell numbers are required to produce measurable signal changes. The cell numbers are cell-type dependent. Highly glycolytic adherent cells must be trypsinised and concentrated prior to measurement. 6 3.3. Data Analysis For semi-quantitative analysis, the rate of signal increase can be used to compare treated and untreated samples or to compile dose response data. 3.4. Monitoring Cell Respiration The ability of the Extracellular O2 Probe assay to assess cell respiration is illustrated in Fig. 2. Dilutions curves for HepG2 cells (Fig. 1A) and primary rat hepatocytes (Fig. 1B) are presented. Fig. 1: Cell dilutions measured on 96-well plates using Extracellular O2 Probe. A) HepG2 cell dilution after an overnight (□) or 2 day culture period (■), B) primary rat hepatocyte cell dilution after an overnight culture period. Rates of probe signal change (slope of fluorescence signal) were normalised against initial intensity. 7 3.5. Extracellular O2 probe and ATP analysis of cells To contrast the sensitivities of oxygen consumption and cellular ATP concentrations as indicators of mitochondrial dysfunction; HepG2 cells were treated with a panel of classical electron transport chain (ETC) modulators and both cellular oxygen consumption and cellular ATP concentrations were measured. 8 Fig 2: Parallel analysis of classical inhibitors on HepG2 cells. A) Extracellular O2 Probe assessment of mitochondrial function immediately post treatment, B) analysis of ATP concentrations 24 h post-treatment. For Extracellular O2 Probe measurements, cells were plated at 80,000 cells/well, allowed to adhere overnight and then assayed. Extracellular O2 data presented in Fig. 2A illustrates that drug induced mitochondrial dysfunction is evident immediately post treatment with both inhibition (Oligomycin, Rotenone, Antimycin) and uncoupling (FCCP) being detected. Despite this dysfunction and an additional 24 hour exposure, analysis of cellular ATP concentrations indicated high levels of ‘viability’ (Fig. 2B). This pattern is also reflected using other assays and cell lines. 3.6. Extracellular O2 Probe: comparison with other methods of cell screening The dose-response relationship for the ETC inhibitor rotenone using four different viability assays: Extracellular O2 probe, Bioluminescent ATP Measurement, LDH and Cellular DNA Measurement outlined in Fig. 3. These data indicate that treatment suppresses mitochondrial respiration, leading to loss of function and reduced oxygen consumption prior to cell death. 9 Fig. 3: Effects of Rotenone treatment on H-4-II-E cells analysed using Extracellular O2 probe, Bioluminescent ATP Measurement, LDH and Cellular DNA Measurement (24h exposure). These results demonstrate the effectiveness of Extracellular O2 Probe in assessing mitochondrial function in whole cells as well as the importance of evaluating mitochondrial function in the context of drug toxicity. The specificity and sensitivity of the Extracellular O2 Probe assay is demonstrated by its ability to detect mitochondrial toxicity more rapidly and at lower drug doses than other assays. When combined with other assays, Extracellular O2 Probe allows detailed evaluation of mechanisms of drug toxicity, adding significantly to the portfolio of information available for compound evaluation. 10 4. Protocol for Plate Based Analysis of Isolated Mitochondria Assay Summary Add Cells, Extracellular O2 Probe +/- ADP and Compound to 96-well plate Add mineral oil to wells Read plate using fluorescence plate reader 4.1. Plate Preparation 4.1.1. Equilibrate plate temperature on a plate heater to 30°C. 4.1.2. Reconstitute Extracellular O2 Probe in 1 ml H2O to give a 1 µM stock solution. Dilute 1:10 in measurement buffer (250 mM sucrose, 15 mM KCl, 1 mM EGTA, 5 mM MgCl2, 30 mM K2HPO4, pH 7.4) and add 100 µl to each well. 4.1.3. For compound testing, add 1 µL of compound in an appropriate solvent to test wells. 4.1.4. Dilute Mitochondria to the desired concentration in measurement buffer (250 mM sucrose, 15 mM KCl, 1 mM EGTA, 5 mM MgCl2, 30 mM K2HPO4, pH 7.4) and add 50 µL to test well. Dissolve substrate (succinate or glutamate/malate) and ADP in 11 measurement buffer and add 50 µL of this solution to test wells giving a final substrate concentration of 25 Mm (succinate) or 12.5/12.5 mM (glutamate/malate) and a final ADP concentration of 1.65 mM. 4.1.5. Add 100 µL of mineral oil (preheated to 30°C) to each well. This increases assay sensitivity by minimising interference from ambient oxygen. Note: Plate preparation time should be kept to a minimum. 4.2. Measurement 4.2.1. Insert the prepared plate into a fluorescence plate reader preset to 30°C. 4.2.2. Measure probe signal at 1.5 min intervals for 10-30 min using excitation and emission wavelengths of 380 nm and 650 nm respectively. (Readers equipped with a time-resolved mode, may achieve improved performance using delay and gate time of 30 and 100 µsec respectively). Note: Mitochondria are freshly prepared as per user’s protocol and should not be left on ice for longer than recommended in the literature. Measurement buffers should be prepared freshly on the day of measurement. 12 4.3. Data Analysis • For semi-quantitative analysis the rate of signal increase can be used to compare treated and untreated samples or to compile dose response data. • For more quantitative analysis, fluorescence data may be related to oxygen concentrations. 5. Assay Throughput and Performance The data output from the Extracellular O2 Probe assay (96 samples), and polarographic (one sample) analysis of mitochondrial oxygen consumption is contrasted in Fig. 4. Fig 4a shows typical polarographic analysis illustrating initiation of State 2 and State 3 respiration through addition of substrate and ADP respectively. Fig 1b shows oxygen consumption measured using Extracellular O2 Probe, with glutamate/malate- (left) and succinate (right) -driven oxygen consumption is measured at decreasing mitochondrial protein concentrations in both State 2 (top) and State 3 (bottom) (n = 4). 13 Fig. 4: Analysis of isolated mitochondrial using A) conventional polarography and B) Extracellular O2 Probe. C) Data extracted from Fig. 4B illustrating activation of succinate driven mitochondrial measured using Extracellular oxygen O2 consumption Probe A65N (n = 4, % CV < 3 %). 14 The compatibility of the Extracellular O2 Probe assay with the microplate format permits analysis under 96 (or 384) discrete conditions. The effectiveness of this level of throughput in analysing isolated mitochondria is highlighted in Fig. 2B which examines increasing mitochondrial protein concentrations on glutamate/malate- and succinate-driven respiration in both basal (State 2) and ADPactivated (State 3) states (all in quadruplicates). The performance of the Extracellular O2 Probe assay is highlighted in Fig. 4C, with coefficient of variance below 3%. 6. Assessment of Classical Mitochondrial Effectors Validation of Extracellular O2 Probe for assessment of mitochondria is illustrated in Fig. 5. These data illustrate the inhibition of mitochondrial function using a panel of classical mitochondrial inhibitors and highlight the dose dependence of this inhibition for KCN. 15 Fig. 5: A) Monitoring the effect of a panel of classical ETC inhibitors on mitochondrial function using Extracellular O2 probe, and B) dose dependent inhibition of mitochondrial function by KCN. 16 7. Compound Screening Extracellular O2 Probe allows screening of compounds at multiple concentrations and in multiple conditions in a single microtitre plate as illustrated in Fig. 6. Such data may be processed further to generate dose response data. Fig. 6: Effect of test compounds (D01-D05), on both State 2 and State 3 isolated mitochondrial function using Extracellular O2 probe. Some compounds uncouple in a dose dependant manner while others inhibit. 17 Overall, Extracellular O2 Probe allows highly sensitive highthroughput detection of mitochondrial dysfunction in isolated mitochondria. Use of a 96-well plate format allows screening of 200 compounds per day at a single dose, or acquisition of dose response characteristics for 25 compounds per day. This capability represents a fundamental shift in the capacity for mitochondrial toxicity testing in drug discovery programs, without compromising data quality or information content. 18 UK, EU and ROW Email: [email protected] Tel: +44 (0)1223 696000 www.abcam.com US, Canada and Latin America Email: [email protected] Tel: 888-77-ABCAM (22226) www.abcam.com China and Asia Pacific Email: [email protected] Tel: 108008523689 (中國聯通) www.abcam.cn Japan Email: [email protected] Tel: +81-(0)3-6231-0940 www.abcam.co.jp 19 Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.