ab Luminescent ATP Detection Assay Kit

Transcription

1 ab Luminescent ATP Detection Assay Kit Instructions for use: For quantitative or qualitative measurement of ATP in cells. This product is for research use only and is not intended for diagnostic use. Version 5 Last Updated 23 August 2016

2 Table of Contents INTRODUCTION 1 1. BACKGROUND 1 2. ASSAY SUMMARY 3 GENERAL INFORMATION 4 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. LIMITATIONS 5 6. MATERIALS SUPPLIED 5 7. MATERIALS REQUIRED, NOT SUPPLIED 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 8 9. REAGENT PREPARATION STANDARD PREPARATION 9 ASSAY PROCEDURE ASSAY PROCEDURE 10 DATA ANALYSIS CALCULATIONS TYPICAL DATA 14 RESOURCES QUICK ASSAY PROCEDURE TROUBLESHOOTING INTERFERENCES FAQS NOTES 20

3 INTRODUCTION INTRODUCTION 1. BACKGROUND Abcam s Luminescent ATP Detection Assay Kit (ab113849) is an ATP monitoring system based on firefly (Photinus pyralis) luciferase, used to determine total levels of cellular ATP in culture. The ATP assay is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. The emitted light is proportional to the ATP concentration inside the cell. The reaction can be summarized as follows: ATP + D-Luciferin + O 2 Oxyluciferin + AMP + PPi + CO 2 + Light This kit irreversibly inactivates ATPases (ATP degrading enzymes) during the lysis step, ensuring that the luminescent signal obtained truly corresponds to the endogenous levels of ATP. The major advantages of this assay are: LUCIFERASE Mg 2+ Long luminescence signal: the half-life of the luminescent signal is greater than 5 hours. Therefore, special luminometer with injectors is not required. Fast: results are obtained in less than 30 minutes. Simple: there are no separation steps and there are only 2 reagent additions. Homogeneous: No cell-harvesting or centrifugation required. Sensitive: detection limit is 5 cells in 100 µl. Wide linear dynamic range: approximately from 0.1 nm to 1 µm of ATP (depending on instrument sensitivity) ab Luminescent ATP Detection Assay Kit

4 INTRODUCTION Total levels of cellular ATP can be used to assess cell viability, cell proliferation and cytotoxicity of a wide range of compounds and biological response modifiers. However, this is not representative of the mitochondrial bioenergetics state of the cell. Cultured cells can survive in the absence of an active electron transport chain by supplying all their ATP demands through glycolysis. Nevertheless, when cells are cultured with non-fermentable carbon substrates such as galactose, they require an active electron transport chain (ETC) to supply all their ATP demand. Under this conditions, damage to the mitochondrial bioenergetics state of the cell will lead to depletion of cellular ATP, which can be easily measured by luminescence as a decrease in total levels of ATP measured by this product. ab Luminescent ATP Detection Assay Kit

5 INTRODUCTION 2. ASSAY SUMMARY Grow cells in 96 well plate (100 µl/well) Treat cells with biological modifiers Add 50 µl detergent and incubate 5 minutes Add 50 µl substrate solution and incubate 5 minutes Measure luminescence ab Luminescent ATP Detection Assay Kit

6 GENERAL INFORMATION 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. We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet. Reagents should be treated as possible mutagens and should be handle with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components. Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipet by mouth. Do not eat, drink or smoke in the laboratory areas. All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures. 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 the Materials Supplied section. Aliquot components in working volumes before storing at the recommended temperature. ab Luminescent ATP Detection Assay Kit 4

7 GENERAL INFORMATION 5. 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. 6. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) Detergent 20 ml 4 C 4 C Substrate Buffer 20 ml 4 C 4 C Substrate (lyophilized) 3 vials 4 C -20 C ATP standard (lyophilized) 1 vial 4 C -20 C ab Luminescent ATP Detection Assay Kit 5

8 GENERAL INFORMATION 7. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: Microplate reader capable of measuring luminescence MilliQ water or other type of double distilled water (ddh 2 O) Pipettes and pipette tips, including multi-channel pipette Assorted glassware for the preparation of reagents and buffer solutions ensure they are ATP-free Tubes for the preparation of reagents and buffer solutions ensure they are ATP-free Sterile 96 well plate with clear flat bottom, preferably white General tissue culture supplies Orbital shaker ab Luminescent ATP Detection Assay Kit 6

9 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. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures. 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. Ensure all reagents and solutions are at the appropriate temperature before starting the assay. Samples which generate values that are greater than the most concentrated standard should be further diluted in the appropriate sample dilution buffer. Make sure you have the right type of plate for your detection method of choice. Make sure all necessary equipment is switched on and set at the appropriate temperature. Work in subdued lighting, out of direct sunlight or direct bright fluorescent lighting. Bright light may cause plate phosphorescence resulting in higher background levels. Phosphorescence has a half-life of several minutes. ab Luminescent ATP Detection Assay Kit 7

10 ASSAY PREPARATION ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening Detergent: Ready to use as supplied. Equilibrate to room temperature before use. Store at 4 C in the dark Substrate Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at 4 C in the dark Substrate: Reconstitute each vial of lyophilized substrate in 5 ml of Substrate Buffer (section 9.2). Once reconstituted, substrate activity will decline to 30% of original levels after 1 week of storage at 4 C. Aliquot reconstituted substrate solution so that you have enough volume to perform the desired number of assays. Store at -20 C in the dark. After thawing, crystals may appear in the buffer. These crystals can be dissolved by warming the vial at room temperature and mixing the contents ATP standard: Amount of ATP standard is lot-specific and it will be specified in the product label. Reconstitute ATP standard in ddh 2 O to make a 10 mm stock solution (i.e., if the amount printed on the label is 12 µmol add 1,200 µl of ddh 2 O). Vortex the vial for one minute until fully dissolved. Aliquot reconstituted ATP standard so that you have enough volume to perform the desired number of assays. Store at -20 C in the dark. ab Luminescent ATP Detection Assay Kit 8

11 ASSAY PREPARATION 10.STANDARD PREPARATION If quantification of ATP levels is not required, then you do not need to prepare the ATP standard. Always prepare a fresh set of standards for every use, if it is necessary to quantify the levels of ATP present in your sample. Discard the working standard dilutions after use as they do not store well (standard solutions are stable up to 8 hours when stored on ice). NOTE: The standard dilution described below is a guideline standard and can be adapted if necessary to fit user s need. If your experiment does not need quantification, jus skip this step Prepare a 100 µm ATP standard by diluting 5 µl of the ATP stock standard (section 9.4) into 495 µl Substrate Buffer Using the 100 µm ATP standard, prepare a standard curve dilution as described in the table below in either a microplate or microcentrifuge tubes: Standard # Sample too dilute Volume of Standard (µl) Assay Buffer (µl) End Conc ATP in well µm µm 2 Std # µm 3 Std # µm 4 Std # nm 5 Std # nm 6 Std # nm 7 None nm ab Luminescent ATP Detection Assay Kit 9

12 ASSAY PROCEDURE ASSAY PROCEDURE 11.ASSAY PROCEDURE Equilibrate all materials and prepared reagents to correct temperature prior to use. We recommended to assay all standards, controls and samples in duplicate. Prepare all reagents, working standards, and samples as directed in the previous sections. We recommend growing the cells directly on the plate in which the assay will be read for simplicity. If this is not possible, you can transfer cell lysate into the reading plate before the read out Grow cells When planning the layout of the assay, please consider the following: Blank wells = include blank wells (without cells) to determine background luminescence levels. Positive control wells = include a positive control to determine normal signal expected (e.g. untreated or vehicle treated culture). Standard curve = if necessary for interpretation of results, ensure that you leave enough empty wells on the plate to add the ATP standard. Figure 1 (below) shows a suggested assay layout template to screen dose response for 3 compounds in triplicate. In this example assay template, rows A and H as well as columns1 and 12 are not seeded with cells and are left with media only. This will help determine the background luminescence. Columns 1 and 12 will be used to set up the ATP standard dilution series. Wells in column 2 are used as positive control: diluent control wells to determine the maximal expected signal in the absence of compound. ab Luminescent ATP Detection Assay Kit 10

13 ASSAY PROCEDURE Figure 1: Suggested assay layout template In a sterile white 96-well plate, grow cells in 100 µl/well at the optimal seeding concentration (see table below for suggested cell line specific seeding numbers). If working with other cell lines, you need to determine in advance the optimal seeding amount by cell titration. HeLa HepG2 HdFn SH-SY5Y Cell Line Seeding/well 12,000 cells/well 25,000 cells/well 12,000 cells/well 50,000 cells/well Treat cells with appropriate growth factors, cytotoxic agents or other type of biological modifiers as desired. ab Luminescent ATP Detection Assay Kit 11

14 ASSAY PROCEDURE ATP detection procedure: If it is necessary to quantify the levels of ATP in your sample, then, pipette 100 µl of complete culture media into empty wells in wells of the plate that have been allocated for the ATP standard dilutions. If you are not using the ATP standard, then proceed to section Add 50 µl of detergent to the 100 µl media in each well Shake the plate for 5 minutes in an orbital shaker at 700 rpm. This step lyses the cells and stabilizes the ATP If using, add 10 µl of the ATP standard dilution series to the appropriate wells and shake the whole plate for 5 minutes an orbital shaker at 700 rpm. If you are not using the ATP standard, then proceed to section Add 50 µl of reconstituted substrate solution (section 9.3) to each of the wells Shake the microplate for 5 minutes in an orbital shaker at 700 rpm. NOTE: Work with subdued lighting, out of direct sunlight or direct bright fluorescent lighting. Bright light may cause plate phosphorescence resulting in higher background levels. Phosphorescence has a half-life of several minutes Dark adapt the plate for 10 minutes Measure luminescence. ab Luminescent ATP Detection Assay Kit 12

15 DATA ANALYSIS DATA ANALYSIS 12.CALCULATIONS If using the ATP standard to quantify signal, samples producing signals greater than that of the highest standard should be further diluted in appropriate buffer and reanalysed, then multiply the concentration found by the appropriate dilution factor Assay with ATP standard: Average the duplicate reading for each standard and sample Subtract the mean luminescence of the blank (Standard #7) from all standard and sample reading. This is the corrected luminescence Plot the corrected luminescence values for each standard as a function of the final concentration of ATP Draw the best smooth curve through these points to construct the standard curve. Calculate the trend line equation based on your standard curve data (use the equation that provides the most accurate fit) Assay without ATP standard: Average the duplicate reading for each sample Subtract background (empty wells) from each sample measurement Determine ATP levels as percentage from positive control. ab Luminescent ATP Detection Assay Kit 13

16 DATA ANALYSIS 13. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed Figure 2: ATP standard curve on opaque white plate. Background-subtracted data values (mean +/- SD) are graphed. 20,000 Luminescent counts 15,000 10,000 5, M Rotenone Vehicle control Figure 3: Rotenone induces cytotoxicity in HepG2 cells. 2.5 x 10 4 HepG2 cells were seeded into each well, allowed to adhere and treat for 4 hours with 25 µm rotenone and vehicle control (DMSO) in glucose-based complete media. After treatment, cells were lysed, exposed to the ATP substrate solution and signal was measured on a luminometer. Mean and standard deviation is plotted for 3 replicates from each condition. ab Luminescent ATP Detection Assay Kit 14

17 DATA ANALYSIS Figure 4: 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) [white bars] and Glycolysis Assay [Extracellular acidification] (ab197244), [black bars] 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) [stripped bars]. ab Luminescent ATP Detection Assay Kit 15

18 RESOURCES RESOURCES 14. QUICK ASSAY PROCEDURE NOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time. Prepare standard, positive control (if using); get equipment ready. Prepare a 96 well plate with cells and biological modifiers in a total volume of 100 µl. If quantifying signal, prepare ATP standard dilution [0.1 nm 10 µm]. Add 50 µl detergent to samples and standards, and incubate 5 minutes in an orbital shaker at 700 rpm. Add 50 µl of substrate solution, and incubate 5 minutes in an orbital shaker at 700 rpm. Read 96 well plate in a luminometer. ab Luminescent ATP Detection Assay Kit 16

19 RESOURCES 15.TROUBLESHOOTING Problem Cause Solution Assay not working Sample with erratic readings Lower/ Higher readings in samples and Standards Use of ice-cold buffer Plate read at incorrect wavelength Use of a different 96- well plate Samples not deproteinized (if indicated on protocol) Cells/tissue samples not homogenized completely Samples used after multiple free/ thaw cycles Use of old or inappropriately stored samples Presence of interfering substance in the sample Improperly thawed components Allowing reagents to sit for extended times on ice Incorrect incubation times or temperatures Buffers must be at room temperature Check the wavelength and filter settings of instrument Colorimetric: Clear plates Fluorometric: black wells/clear bottom plate Use provided protocol for deproteinization Use Dounce homogenizer, increase number of strokes Aliquot and freeze samples if needed to use multiple times Use fresh samples or store at - 80 C (after snap freeze in liquid nitrogen) till use Check protocol for interfering substances; deproteinize samples Thaw all components completely and mix gently before use Always thaw and prepare fresh reaction mix before use Verify correct incubation times and temperatures in protocol ab Luminescent ATP Detection Assay Kit 17

20 RESOURCES Problem Cause Solution Standard readings do not follow a linear pattern Unanticipated results Pipetting errors in standard or reaction mix Air bubbles formed in well Standard stock is at incorrect concentration Measured at incorrect wavelength Samples contain interfering substances Sample readings above/ below the linear range Avoid pipetting small volumes (< 5 µl) and prepare a master mix whenever possible Pipette gently against the wall of the tubes Always refer to dilutions described in the protocol Check equipment and filter setting Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range ab Luminescent ATP Detection Assay Kit 18

21 RESOURCES 16. INTERFERENCES These chemicals or biological materials will cause interference in this assay causing compromised results or complete failure: Anionic detergents such as SDS: anionic detergents will inhibit the luciferase reaction EDTA will inhibit luciferase reaction it is a quelating agent and magnesium is needed for the reaction Monovalent metal salts such as KCl (concentrations > than 1µM) will affect luciferase activity High concentration of divalent metals such as magnesium (> 3 mm), manganese (> 2 mm) or calcium (> 1 mm) will affect luciferase activity 17. FAQs Can I standardize the results by protein quantification? The mammalian cell lysis solution used in the assay is alkaline and not compatible with protein quantification methods. Can I pause the experiment at any moment? Although the signal of luciferase is long lasting, there will be a small decay over 5 hours (15 20% from original signal). Theoretically, the plate could be stored in the fridge for a very short period of time (e.g. lunch break, but not overnight) after cell lysis. ab Luminescent ATP Detection Assay Kit 19

22 RESOURCES 18. NOTES ab Luminescent ATP Detection Assay Kit 20

23 RESOURCES ab Luminescent ATP Detection Assay Kit 21

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