User Manual- ENZ-51019-KP002 Rev 2.1.1 September 2010.pub
Contents
1. 00 00 oOo x ooo LEID God Doo000 D 0000 D ooo See aoc Enzo Enabling Discovery in Life Science Mito ID Membrane Potential Cytotoxicity Kit for microplates Instruction Manual Cat ENZ 51019 KP002 for 2 x 96 well plates For research use only Rev 2 1 1 September 2010 Notice to Purchaser The Mito ID Membrane Potential Cytotoxicity Kit is a member of the CELLestial product line reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications CELLestial reagents and kits are optimal for use in demanding cell analysis applications involving confocal micros copy flow cytometry microplate readers and HCS HTS where consistency and repro ducibility are required This product is manufactured and sold by ENZO LIFE SCIENCES INC for research use only by the end user in the research market and is not intended for diagnostic or therapeutic use Purchase does not include any right or license to use develop or otherwise exploit this product commercially commercial use development or exploitation of this product or development using this product without the express prior written authorization of ENZO LIFE SCIENCES INC is strictly prohibited Limited Warranty These products are offered under a limited warranty The products are guaranteed to meet appropriate2. accumula tion in the mitochondria contributes to the toxicity of various organs espe cially the heart and liver Adverse drug reactions often remain undetected until large numbers of patients have already been exposed The ability to test a compound s safety at the stage of lead drug selection 10 100 compounds would provide a valuable tool for drug screening research Enzo Life Sciences Mito ID Membrane Potential Cytotoxicity Kit meas ures mitochondrial membrane potential with a cationic dye that fluoresces either green or orange depending upon mitochondrial membrane potential status In energized cells the Mito ID membrane potential dye exists as a green fluorescent monomer in the cytosol and also accumulates as orange fluorescent aggregates in the mitochondria However in cells with compromised mitochondrial membrane potential the Mito ID membrane potential dye exists primarily as green fluorescent monomers throughout the cytosol and no longer exhibits orange fluorescence in the mitochondria Compared with the commonly used cationic carbocyanine dye JC 1 the Mito ID membrane potential dye offers greater solubility better photosta bility and higher sensitivity to mitochondrial membrane potential changes A control mitochondrial membrane potential perturbation agent carbonyl cyanide 3 chlorophenylhydrazone CCCP is provided for monitoring changes in mitochondrial dynamics The Mito ID Membrane Potential Cytotoxicity Kit enab
3. of the mitochondria in particular of the overall function of the respiratory chain and the potential of the mitochondria to generate ATP and provide energy for other cellular components Measurement of the mitochondrial membrane potential is useful in a wide variety of research areas and mitochondrial dysfunction is implicated in diseases such as cancer diabetes Parkinson s disease and stroke Typical results of fluorescence microplate based analysis of cell populations using the Mito ID Membrane Potential Cytotoxicity Kit are shown in Figure 1 Using a conventional fluorescence microplate reader mitochondrial membrane potential is shown to decrease as a function of CCCP concentration as demonstrated by a decrease in orange signal high Z factor 20 9 obtained using this kit demonstrates excellent signal to background ratios Figure 2 shows real time kinetic measurement of mitochondrial mem brane potential changes with CCCP treatment using a conventional fluorescence microplate reader with liquid dispensing capability for test agent addition The Mito ID MP dye provides a more robust method to monitor mito chondrial membrane potential changes compared to JC 1 due to its higher photostability better aqueous solubility and higher sensitivity to mitochondrial membrane potential changes This kit offers true mix and read capability and can be readily used for screening of large compound libraries Enzo Kit Z factor 0 90
4. Control cells are not healthy Extended storage of cells after staining may adversely affect their health Use healthy cells Acquire data as soon as possible after staining the cells Cell staining is too strong Mitochondrial membrane potential dye is too concen trated for the cell type being employed Dilute the staining solution further with Assay Buffer Orange fluorescent mito chondrial membrane poten tial signal increases upon treatment with the CCCP Control The orange emission filter in your instrument may be too wide and green fluorescent signal is being read too Replace emission filter with a narrower band pass filter Accuracy of data from experiment to experiment is poor The concentration of the stained cell sample for data acquisition is too low or inconsistent between wells Repeat experiments using a higher density of cells and carefully seed the wells so that each well has the same number of cells 10 Enzo Life Sciences www enzolifesciences com Enabling Discovery in Life Science NORTH SOUTH AMERICA ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA T 1 800 942 0430 610 941 0430 F 610 941 9252 E info usaGenzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 441 061 926 89 89 F 441 061 926 89 79 E i
5. JC 1 Z factor 0 60 JC 1 Kit Mito ID Membrane Potential Cytotoxicity Kit g E i a v 5 5 1 10 CCCP concentration uM Figure 1 Using a conventional fluorescence microplate reader mitochondrial membrane potential was shown to decrease as a function of CCCP concentration as demonstrated by a decrease in orange signal Enzo s Mito ID MP dye is at least 10 fold more sensitive to MMP loss than JC 1 The high Z factor 20 9 obtained using the Enzo kit demon strates excellent signal to noise and signal to background ratios The error bars denote the standard deviation of at least eight determinations Treated Control of Mito ID MP Orange Signal Change 7 8 9 10 11 12 13 14 15 Time Min Figure 2 Real time kinetic study of mitochondrial membrane potential changes arising from CCCP treatment Data were collected using a conventional fluorescence microplate reader with liquid dispensing capability Hela cells were seeded overnight 25 000 cells per 100 ul per well in 96 well black wall clear bottom plate Next day 100 ul of Mito ID MP dye was directly added into each well and cells were incubated for 30 mins at room temperature CCCP 20 ul well was added using a BioTek dual liquid dispensor to achieve concentrations of 1 Orange Mito ID MP dye signal was shown to change in the presence of CCCP as a function of time as demonstrated by a decrease in signal read using the Bio
6. Tek Synergy Mx fluorescence microplate reader VII References 1 Smiley Reers Mottola Hartshorn Lin Chen Smith Steele and Chen 1991 Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J aggregate forming lipophilic cation JC 1 Proc Natl Acad Sci USA 88 3671 3675 Cossarizza Baccarani Contri Kalashnikova and Franceschi 1993 A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J aggregate forming lipophilic cation 5 5 6 6 tetrachloro 1 1 3 3 tetraethylbenzimidazolylcarbocyanine iodide JC 1 Biochem Biophys Res Commun 197 1 40 45 Woollacott and Simpson 2001 High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells J Biomol Screen 6 6 413 420 VIII Troubleshooting Guide Problem Potential Cause Suggestion Fixed and or permeabilized cells fail to stain with the mitochondrial membrane potential dye This dye is only suitable for live cell staining Use the dye only for live cell analysis Poor staining observed Stained cells have been exposed to strong light Protect samples from expo sure to strong light and analyze them immediately after staining Kit reagent has degraded Verify that the reagents are not past their expiration dates before using them Control cells without treatment show low orange signal
7. ects of the compound For positive control dispense 10 20uL of provided Control to a final concentration of 2 4 uM in medium or buffer for 15 30 minutes Negative control cells should be treated with the same vehicle media or buffer used to reconstitute or dilute the inducer or inhibitor for an equal length of time under similar conditions NOTE It is NOT necessary to remove medium or wash the cells However some media may result in high background levels and some test compounds may be sensitive to the presence of serum In these instances growth media and serum should be removed Then add 100 uL of the buffer of choice to the wells before adding the dye loading solution 5 NOTE Titration of the CCCP Control may be required for optimal results with different cell types 3 Dispense 100 uL of the prepared Mito ID MP Dye Loading Solution see section B 2 page 4 into each well Incubate for 30 minutes at room temperature or 37 C NOTE The Mito ID MP aye is light sensitive Be sure to pro tect samples from light The appropriate incubation time depends upon the individual cell type and cell concentration used Optimize the incubation time for each experiment 4 Observe immediately with a fluorescent microplate reader or imaging plate reader using an excitation setting of about 480 nm and an emission setting of about 590 nm In live and healthy cells the mitochondria will fluoresce orange following aggregation
8. id direct exposure of the reagent to intense light Aliquot and store unused reagent at 20 C protected from light Avoid repeated freeze thaw cycles For each 96 well plate add 100 uL of Mito ID MP Detection Re agent 1 mL 10X Assay Buffer 1 and 0 2 mL 50X Assay Buffer 2 into 8 7 mL deionized water Mix well C MEMBRANE POTENTIAL ASSAYS Rapid Kinetics Measurement for compounds that change mitochondrial membrane potential within 30 mins 1 Obtain prepared microplates containing cells see section A 2 Dispense 100 uL of the prepared Mito ID MP Dye Loading Solution see section B 2 above directly into the growth medium NOTE It is NOT necessary to remove medium or wash the cells However some media may result in high background levels and some test compounds may be sensitive to the presence of serum In these instances growth media and serum should be removed Then add 100 uL of the buffer of choice to the wells before adding the dye loading solution 3 Incubate the microplates for 30 mins at room temperature or 37 C prior to addition of the test compound NOTE The Mito ID MP dye is light sensitive Be sure to protect samples from light The appropriate incubation time depends upon the individual cell type and cell concentration used Optimize the incubation time for each experiment DO NOT wash the cells after dye loading Prepare the test compound by dissolving it in the buffer
9. les monitoring of mitochondrial potential changes using a simple fluorescence microplate reader Potential applications of the kits are in preclinical drug safety assessment using in vitro cell culture models to aid in the drug develop ment process and especially to differentiate among test compounds and rank their order of potency Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored upright and protected from light at s 20 C When stored properly these reagents are stable for at least twelve months Avoid repeated freezing and thawing The reagents provided in the kit are sufficient for 2 x 96 well microplates Reagent Quantity Mito ID MP Detection Reagent 200 uL CCCP Control 2 mM 100 uL 10X Assay Buffer 1 2 5 mL 50X Assay Buffer 2 0 5 mL Additional Materials Required Fluorescence microplate reader or imaging plate reader with a filter set or monochromator setting that provides 490 570 590 nm 96 well tissue culture microplate with black wall and clear bottom e Calibrated adjustable precision pipetters preferably with disposable plastic tips Glass microscope slides optional Glass cover slips optional Deionized water Anhydrous DMSO optional e Serum optional Growth medium e g Dulbecco s modified Eagle medium D MEM IV Safety Warnings and Precautions This product is for research use only and is not inte
10. nded for diagnostic purposes Some components of this kit may contain hazardous substances Reagents can be harmful if ingested or absorbed through the skin and may cause irritation to the eyes Reagents should be treated as possible mutagens and should be handled with care and disposed of properly 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 blood components and biological materials should be treated as potentialy hazardous and handled as such They should be disposed of in accordance with established safety procedures To avoid photobleaching perform all manipulations in subdued light environments in amber microcentrifuge tubes or protected from light by other means Methods and Procedures A CELL PREPARATION Cells should be maintained in 96 well microplates via standard tissue culture practices Seed cells in microplate s the day before test compound probe addition The protocols described herein are based upon Hela cells for which it is highly recommended that cells in culture medium are seeded on plates at a density of 2 x 10 to 3 10 cells per well at a seeding volume of 100 uL per well Keep the microplate s in a humidified CO incubator overnight NOTE 96 well black wall and clear bottom plates are highly recom mended for this assay Any cell number and plate coating requirements sh
11. nfo chGenzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 0 7621 5500 526 Toll Free 0800 664 9518 F 449 0 7621 5500 527 E info deGenzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 32 03 466 04 20 432 03 466 0429 E info be enzolifesciences com www enzolifesciences com incorporating NTERNA TONAL UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 0845601 1488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info uk enzolifesciences com www enzolifesciences com www enzolifesciences com FRANCE ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert Einstein FR 69100 Villeurbanne France T 33 472 440 655 F 33 437 484 239 E info fr enzolifesciences com www enzolifesciences com Alexis assay designs Stressgen
12. of choice The Mito ID Membrane Potential Cytotoxicity Assay is optimized for compound addition at one tenth of the final well volume For positive control dispense 20 uL of the provided Control to a final concentration of 2 4 uM in medium or buffer NOTE Titration of the Control may be required for optimal results with different cell types Run the Mito ID membrane potential cytotoxicity assay by monitoring the fluorescence at 490 nm Em 590 nm with a fluorometric imaging plate reader or a fluorescence microplate reader equipped with liquid dispensing capabilities In live and healthy cells the mitochondria will fluoresce orange following aggregation of the Mito ID MP dye The orange J aggregates emit at 590 nm NOTE Faster addition speeds can lead to better mixing of com pounds and lower signal variance across the plate Make sure to follow the recommended experimental setup parameters provided by the instrument manufacturer before reading the plate It is also important to run the signal test before the experiment Different instruments have their own intensity range Il Slow Kinetics Measurement for compounds that change mitochondrial membrane potential after more than 30 min treatment 1 Obtain prepared microplates containing live cells see section A Treat the cells with 10 20 uL of your compound under optimal culture or buffer conditions for a time period sufficient for assessing the eff
13. of the Mito ID MP dye The orange J aggregates emit at 590 nm NOTE lt is imperative that samples be analyzed immediately following completion of staining DO NOT wash the cells after Mito ID MP dye loading The fluorescence value in blank wells added with the growth medium should be subtracted from the values for those wells with cells treated with the test compounds VI APPENDICES A FiLTER SET AND MONOCHROMATOR SETTING SELECTION The selection of optimal filter sets or monochromator settings for a fluorescence microplate reader application requires matching the in strument optical specifications to the spectral characteristics of the dyes employed in the analysis Please consult your instrument or filter set manufacturer for assistance in selecting optimal filter or mono chromator settings Settings suitable for Texas Red or tetramethylrhodamine TRITC are recommended for imaging the orange fluorescent signal of the Mito ID MP dye indicating energized mitochondria B EXPECTED RESULTS In most eukaryotic cells the majority of ATP production is via oxidative phosphorylation by the mitochondrial respiratory chain Therefore the mitochondrial membrane potential makes up a large part of the bioenergetic state of the cell and it is altered directly depending upon the cell s energy needs The ability to accurately measure mitochon 6 drial membrane potential can give invaluable information about the general health and function
14. ould be optimized for the selected cell REAGENT PREPARATION NOTE Allow all reagents to thaw at room temperature before starting with the procedures Upon thawing gently hand mix or vortex the re agents prior to use to ensure a homogenous solution Briefly centrifuge the vials at the time of first use as well as for all subsequent uses to gather the contents at the bottom of the tube 1 Positive Control CCCP carbonyl cyanide 3 chlorophenylhydrazone is a proton ionophore and uncoupler of oxidative phosphorylation in mitochondria As such it is useful for depolarizing mitochondria Addition of CCCP should cause a dose dependent reduction in mitochondrial orange fluorescence Prepare this perturbation agent as a positive control for mitochondrial membrane potential loss Before beginning the experiment ensure that the vial of CCCP Control has equilibrated to room temperature Dilute the CCCP Control supplied as a 2 mM stock solution in DMSO in medium or buffer of choice to a final concentration optimized for the cells being used For HeLa or U2OS cells it is recommended to perform treatment with the agent using a final assay concentra tion of 2 4 uM CCCP in medium for about 15 30 minutes in order to observe changes in mitochondria membrane potential changes Unused stock should be stored in aliquots at 20 C for about 6 months 2 Mito ID MP Dye Loading Solution NOTE The Mito ID MP Detection Reagent is light sensitive Avo
15. specifications described in the package insert at the time of shipment Enzo Life Sciences sole obligation is to replace the product to the extent of the purchase price All claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo CELLestial and Mito ID are trademarks of Enzo Life Sciences Inc Several of Enzo s products and product applications are covered by US and foreign patents and patents pending Contents VI VII Vill Introduction 1 Reagents Provided and Storage 2 Additional Materials Required 2 Safety Warnings and Precautions 3 Methods and Procedures 3 A CELL 404 40 00 cono 3 REAGENT 4 3 C MEMBRANE POTENTIAL CYTOTOXICITY ASSAYS 4 Appendices eee 6 A FILTER SET 22 2 0044000000 6 RESULTS RSS REDE 6 References daa danos 9 Troubleshooting Guide 10 Introduction Mitochondria play a central role in diverse biological phenomena including metabolism bioenergetics cancer and apoptosis Recently it has been found that compromised membrane potential induced by drug
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