User Manual-ENZ-51022-K500 Rev 1.0.2 April 2011.pub
Contents
1. sequestered by actively respiring mitochondria However these mitochondrial stains are subsequently leached out of cells once the mito chondria s membrane potential dissipates This characteristic severely limits their use in experiments in which cells must be treated with nonionic detergents aldehyde fixatives or other agents that affect the energetic state of the mitochondria Micromolar concentrations of Mito ID Green dye are sufficient for staining mammalian cells This has been validated with a human cervical carcinoma cell line HeLa a human T lymphocyte cell line Jurkat and human bone osteosarcoma epithelial cell line U2OS One important application of Mito ID Green dye is in fluorescence co localization imaging with red fluorescent protein RFP tagged proteins This is a powerful approach for determining the targeting of molecules to intracellular compartments and for screening of associations and interac tions between these molecules Additionally many organelle targeting probes photobleach rapidly are subject to quenching when concentrated in organelles are highly toxic or only transiently associate with the target organelle requiring imaging within a minute or two of dye addition Enzo s Mito ID Green dye a cell permeable small organic probe molecule that spontaneously localizes to live or fixed mitochondria was developed to overcome the above problems The Mito ID Green dye can be readily used in combination with o2. 42 0430 610 941 0430 F 610 941 9252 E info usa enzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 41 0619268989 F 41 0619268979 E info ch Genzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 449 0 7621 5500 526 Toll Free 0800 664 9518 F 49 0 7621 5500 527 E info deGenzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 32 0 3 466 04 20 F 32 0 3 466 04 29 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 433472440655 F 433437 484 239 E info fr enzolifesciences com www enzolifesciences com ALEXIS assay designs Stressgen
3. Enzo Enabling Discovery in Life Science Mito ID Green Detection Kit for fluorescence microscopy Instruction Manual Cat No ENZ 51022 K500 500 assays For research use only Rev 1 0 2 April 2011 Notice to Purchaser The Mito ID Green Detection Kit is a member of the CELLestial product line re agents and assay kits comprising fluorescent molecular probes that have been exten sively benchmarked for live cell analysis applications CELLestial reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy flow cytometry microplate readers and HCS HTS where consistency and reproducibility 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 Any 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 appropriate 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 claim
4. ce the mitochondrial membrane potential is dissipated Mito ID Green dye accumulates in the mitochondria regardless of the mitochondrial membrane potential The dye selectively stains mitochondria of living cells and is relatively insensitive to mitochon drial membrane potential uncouplers of phosphorylation such as CCCP carbonyl cyanide 3 chlorophenylhydrazone as well as ion channel drugs such as valinomycin In addition to being a live cell permeable dye the Mito ID Green dye is functional after cell fixation and detergent permeabilization Mito ID Green dye has been shown to co localize with Mito ID Red dye Previously the Mito ID Red dye has been shown to co localize with mitochondria expressing a mitochondrial GFP tag 6 VII References 1 Freundt Czapiga and Lenardo 2007 Photoconversion of Lysotracker Red to a green fluorescent molecule Cell Res 17 11 956 958 2 Nadrigny Li Kemnitz Ropert Koulakoff Rudolph Vitali Giaume Kirchhoff and Oheim 2007 Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles Biophys J 93 3 969 980 3 Minamikawa Sriratana Williams Bowser Hill and Nagley 1999 Chloromethyl X rosamine MitoTracker Red photosensitises mitochondria and induces apoptosis in intact human cells Journal of Cell Science 112 2419 2430 4 Scorrano Petronilli Colonna Di Lisa and Bernardi 1999 Chloro methyltetramethylrosamine Mitotrac
5. d dilution NOTE If desired standard immunofluorescence staining protocols using coumarin based cyanine 5 or other red or blue fluo rescent antibody conjugates or equivalent should be per formed before Staining with Mito ID Green Antifade compounds or mounting media may be beneficial Try to view the samples as soon as possible after staining for sharper staining VI APPENDICES A Filter Set Selection The selection of optimal filter sets for a fluorescence microscopy application requires matching the optical filter specifications to the spectral characteristics of the dyes employed in the analysis Consult the microscope or filter set manufacturer for assistance in selecting optimal filter sets for your microscope 0 8 07 0 6 0 5 04 03 02 0 1 0 0 300 350 400 450 500 550 600 650 700 350 400 450 500 550 600 Wavelength Wavelength Figure 1 Absorption excitation and fluorescence emission spectra for Mito ID Green Ex Em 460 560 nm panel A and Hoechst 33342 Ex Em 350 461 nm panel B dyes All spectra were determined in 1X Assay Buffer B Results Mitochondria are subcellular organelles found in eukaryotic cells often representing as much as 10 of the total cell volume Although conventional fluorescent stains for mitochondria such as JC 1 rhodamine 123 and tetramethylrhodamine are readily sequestered by functioning mitochondria they are subsequently leached out of the cells on
6. e cells with 100 uL 1X Assay Buffer Remove excess buffer Resuspend cells in 30 uL 1X Assay Buffer then apply the cells to a glass slide and overlay with a coverslip Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC filter set for imaging the mitochondria Optionally image the nucleus using a DAPI filter set and the RFP tagged protein using a Texas Red filter set D STAINING OF ALDEHYDE FIXED AND DETERGENT PERMEABILIZED CELLS The Mito ID Green dye is capable of staining already fixed and permeabilized cells 1 Growth of cells should be performed as described in sections B or C 2 Carefully remove the growth medium or 1X Assay Buffer covering the cells and replace it with freshly prepared medium or buffer containing 3 796 formaldehyde 3 Incubate the cells at 37 C for 15 minutes 4 After fixation wash the cells in PBS or 1X Assay Buffer 5 If the cells are to be subsequently labeled with an antibody a permeabilization step is usually required to enhance the antigen s accessibility Incubate the fixed cells in PBS or 1X Assay Buffer containing 0 196 Triton X 100 at room temperature for about one minute 6 Following permeabilization rinse the cells twice in PBS or 1X Assay Buffer 7 Perform staining as recommended for adherent or suspension cells sections B or C using a 500 fold dilution of the Mito ID Green instead of the 1000 fol
7. fuge with swinging buckets for suspension cultures Glass microscope slides Glass cover slips Deionized water Anhydrous DMSO optional Growth medium e g Dulbecco s Modified Eagle Medium D MEM Formaldehyde optional for fixation protocol Triton X 100 optional for permeabilization protocol Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes The Mito ID Green Detection Reagent contains DMSO which is read ily absorbed through the skin It is harmful if ingested or absorbed through the skin and may cause irritation to the eyes Observe appro priate precautions when handling 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 potentially hazardous and handled as such They should be disposed of in accordance with established safety procedures e To avoid photobleaching perform all manipulations in low light environments or protected from light by other means V Methods and Procedures NOTE Allow all reagents to thaw at room temperature before starting with the procedures Upon thawing gently hand mix or vortex the reagents prior to use to ensure a homogenous s
8. ker Orange Induces the Mito chondrial Permeability Transition and Inhibits Respiratory Complex I Implications for the mechanism of cytochrome c release J Biol Chem 274 35 24657 24663 VIII Troubleshooting Guide Problem Potential Cause Suggestion Mitochondria are not suffi ciently stained Precipitate is seen in the 10X Assay Buffer Very low concentration of Mito ID green dye was used or dye was incubated with the cells for an insuffi cient length of time Precipitate forms at low temperatures Either increase the labeling concentration or increase the time allowed for the dye to accumulate in the mito chondria Allow solution to warm to room temperature or 37 C then vortex to dissolve all precipitate Blue nuclear counterstain is too bright compared to the green mitochondrial stain Different microscopes cameras and filters may make some signals appear very bright Reduce the concentration of the nuclear counterstain or shorten the exposure time Cells do not appear healthy Some cells require serum to remain healthy Add serum to stain and wash solutions Serum does not affect staining Normal amounts of serum added range from 2 to 1096 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 9
9. olution 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 A REAGENT PREPARATION 1 1X Assay Buffer Allow the 10X Assay Buffer to warm to room temperature Make sure that the reagent is free of any crystallization before dilution Prepare enough 1X Assay Buffer for the number of samples to be assayed by diluting each milliliter mL of the 10X Assay Buffer with 9 mL of deionized water Dual Detection Reagent The concentration of Mito ID Green dye for optimal staining will vary depending upon the application Suggestions are provided to use as guidelines though some modifications may be required depending upon the particular cell type employed and other factors such as the permeability of the dye to the cells or tissues To reduce potential artifacts from overloading of the cells the concentration of the dye should be kept as low as possible Prepare sufficient amount of Dual Detection Reagent for the number of samples to be assayed as follows For every 1 mL of 1X Assay Buffer see preparation in step 1 or cell culture medium add 1 uL of Mito ID Green Detection Reagent and 1 uL of Hoechst 33342 Nuclear Stain NOTE a The dyes may be combined into one staining solution or each may be used separately if desired b The Hoechst 33342 Nuclear Stain can be diluted further if its staining intensity is much stronger than the green Mitoch
10. ondrial stain Mito ID Green c When staining BFP or CFP expressing cells the Hoechst 33342 Nuclear Stain should be omitted due to its spectral overlap with these fluorescent proteins B STAINING LIVE ADHERENT CELLS 1 Grow cells on cover slips inside a Petri dish filled with the appro priate culture medium When the cells have reached the desired level of confluence carefully remove the medium Dispense sufficient volume of Dual Detection Reagent see section V A2 page 3 to cover the monolayer cells 100 uL of labeling solution for cells grown on an 18 X 18 mm coverslip Protect samples from light and incubate for 15 30 minutes at 37 C Optional Wash the cells with 100 uL 1X Assay Buffer Remove excess buffer and place coverslip on slide Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC filter set for imaging the mitochondria Optionally image the nucleus using a DAPI filter set and the RFP tagged protein using a Texas Red filter set C STAINING LIVE CELLS GROWN IN SUSPENSION 1 Centrifuge cells for 5 minutes at 400 x g at room temperature RT to obtain a cell pellet Carefully remove the supernatant by aspiration and dispense sufficient volume of Dual Detection Reagent see section V A2 page 3 to cover the dispersed cell pellet Protect samples from light and incubate for 15 to 30 minutes at 37 C Optional Wash th
11. s 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 I Introduction eii II Reagents Provided and Storage III Additional Materials Required IV Safety Warnings and Precautions V Methods and Procedures nes A REAGENT PREPARATION eese B STAINING LIVE ADHERENT CELLS C STAINING LIVE CELLS GROWN IN SUSPENSION D STAINING OF ALDEHYDE FIXED AND DETERGENT PERMEABILIZED CELLS VI Appendices nana nenia kaamera iniaa A FILTER SET SELECTION i B RESULTS E E E E VII References niii ae VIII Troubleshooting Guide Introduction Enzo Life Sciences Mito ID Green Detection Kit contains a novel mito chondria selective dye suitable for live detergent permeabilized and alde hyde fixed cell staining Conventional fluorescent stains for mitochondria such as JC 1 Cat No ENZ 52304 rhodamine 123 Cat No ENZ 52307 and tetramethylrhodamine ethyl ester TMRE Cat No ENZ 52309 are readily
12. ther common UV and visible light excitable fluo rescent dyes and various fluorescent proteins in multi color imaging and detection applications It emits in the FITC region of the visible light spec trum and is highly resistant to photobleaching and concentration quench ing The Mito ID Green Detection Kit has been specifically designed for use with RFP expressing cell lines as well as cells expressing blue or cyan fluorescent proteins BFPs and CFPs Additionally the kit is suitable for use with live or fixed cells in conjunction with fluorescent probes such as labeled antibodies or other fluorescent conjugates displaying similar spectral properties as coumarin Texas Red and cyanine 5 A nuclear counterstain Hoechst 33342 is provided to highlight this organelle as well Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored at lt 20 C protected from light When stored properly these reagents are stable for at least twelve months Avoid repeated freezing and thawing Reagents provided in the kit are sufficient for approximately 500 assays using either live adherent cells or cells in suspension Reagent Quantity Mito ID Green Detection Reagent Hoechst 33342 Nuclear Stain 10X Assay Buffer Additional Materials Required Standard fluorescence microscope Calibrated adjustable precision pipetters preferably with disposable plastic tips Adjustable speed centri
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