User Manual-ENZ-51007-500
Contents
1. cells expressing blue cyan or yellow fluorescent proteins BFPs CFPs YFPs Additionally the kit is suitable for use with live or post fixed cells in conjunction with probes such as labeled antibodies or other fluorescent conjugates displaying similar spectral properties as fluorescein or coumarin A nuclear counterstain is provided to highlight this organelle as well 1 II Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored at 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 III Additional Materials Required Standard fluorescence microscope Calibrated adjustable precision pipetters preferably with disposable plastic tips Adjustable speed centrifuge 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 Mito ID Red Antifade Reagent or Mito ID Red Antifade Reagent with DAPI ENZ 53002 M010 or ENZ 53003 M010 optional IV Safety Warnings and Precautions2. Mito ID Red Detection Kit GFP Certified Mitochondrial Detection for microscopy Instruction Manual Cat No ENZ 51007 500 500 assays For research use only Rev 1 2 3 June 2011 Enabling Discovery in Life Science Notice to Purchaser The Mito ID Red Detection Kit GFP Certified Mitochondrial Detection 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 applica tions CELLestial reagents and kits are optimal for use in demanding cell analysis appli cations 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
3. SET SELECTION 6 B RESULTS 7 VII References 8 VIII Troubleshooting Guide 8 I Introduction Enzo Life Sciences Mito ID Red Detection Kit GFP Certified Mitochon drial Detection contains a novel mitochondria selective dye suitable for live detergent permeabilized and aldehyde fixed cell staining Conven tional fluorescent stains for mitochondria such as JC 1 Catalog No ENZ 52304 rhodamine 123 Catalog No ENZ 52307 and tetra methylrhodamine ethyl ester TMRE Catalog No ENZ 52309 are read ily sequestered by actively respiring mitochondria However these mito chondrial 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 Sub micromolar concentrations of Mito ID Red 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 Red dye is in fluorescen
4. This product is for research use only and is not intended for diagnostic purposes The Mito ID Red Detection Reagent contains DMSO which is readily absorbed through the skin It is harmful if ingested or absorbed through the skin and may cause irritation to the eyes Observe appropriate 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 Reagent Quantity Mito ID Red Detection Reagent 10 L Hoechst 33342 Nuclear Stain 50 L 10X Assay Buffer 15 mL 2 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 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 A REAGENT PREPARATION 1 1X Assay Buffer Allow t
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6. 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 1 Triton X 100 at room temperature for no more than 5 minutes 6 Following permeabilization rinse the cells in PBS or 1X Assay Buffer NOTE If desired standard immunofluorescence staining protocols using fluorescein or coumarin based antibody conjugates or equivalent may be performed after fixation and permeabilization steps Antifade compounds using phenylenediamine are not recom mended If an antifade is desired we recommend Enzo s Mito ID Red Antifade Reagent ENZ 53002 M010 or Mito ID Red Antifade Reagent with DAPI ENZ 53003 M010 Try to view the samples as soon as possible after staining for sharper staining 7 If antifade is desired remove all liquid Add one drop of antifade warmed to room temperature then carefully add a coverslip over sample being careful not to introduce air bubbles For sharper images the sample may be viewed immediately or for long term storage the sample may be allowed to dry overnight before sealing the coverslip 5 E STAINING OF ALDEHYDE FIXED AND DETEGENT PERMEABILIZED CELLS The Mito ID Red dye is capable of staining already fixed and permeabilized cells It is not recommended to stain fixed cells with both Mito ID Red dye and th
7. ce co localization imaging with green fluorescent protein GFP 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 However to date photoconversion of red fluorescent dyes to green and metachromatic artifacts wherein fluorescent dyes emit both in the red and green regions of the spectrum have led to spurious results in GFP co localization experiments 1 2 Additionally many organelle targeting probes photobleach rapidly are subject to quenching when concentrated in organelles are highly toxic or only transiently asso ciate with the target organelle requiring imaging within a minute or two of dye addition 3 4 Enzo s Mito ID Red dye a new red emitting cell permeable small organic probe molecule that spontaneously localizes to live or fixed mitochondria was developed to overcome the above problems The Mito ID Red dye can be readily used in combination with other common UV and visible light excitable fluorescent dyes and various fluorescent proteins in multi color imaging and detection applications It emits in the Texas Red region of the visible light spectrum and is highly resistant to photobleaching concentra tion quenching and photoconversion The Mito ID Red Detection Kit GFP Certified Mitochondrial Detection is specifically designed for use with GFP expressing cell lines as well as
8. e Hoechst nuclear counterstain 1 Fixation and permeabilization should be performed as described in section D 2 Perform staining as recommended for adherent or suspension cells sections B or C using a 2 500 fold dilution of the Mito ID Red dye instead of the 10 000 fold dilution NOTE If desired standard immunofluorescence staining protocols using fluorescein or coumarin based antibody conjugates or equivalent should be performed before Staining with Mito ID Red Antifade formulations using phenylenediamine are not recommended If an antifade agent is desired we recommend Enzo s Mito ID Red Antifade Reagent ENZ 53002 M010 or Mito ID Red Antifade Reagent with DAPI ENZ 53003 M010 Try to view the samples as soon as possible after staining for sharper staining 3 If an antifade agent is desired remove all liquid Add one drop of antifade reagent warmed to room temperature then carefully add a coverslip over sample being careful not to introduce air bubbles For sharper images the sample may be viewed immedi ately or for long term storage the sample may be allowed to dry overnight before sealing the coverslip 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 ass
9. he red Mitochondrial stain Mito ID Red d When staining BFP or CFP expressing cells the Hoechst 33342 Nuclear Stain should be omitted due to its spectral overlap with these fluorescent proteins 3 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 2 Dispense sufficient volume of Dual Detection Reagent see section V A2 page 3 to cover the monolayer cells 100 L of labeling solution for cells grown on an 18 X 18 mm coverslip 3 Protect samples from light and incubate for 15 30 minutes at 37 C 4 Wash the cells with 100 L 1X Assay Buffer Remove excess buffer and place coverslip on slide 5 Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard Rhodamine or Texas Red filter set for imaging the mitochondria Optionally image the nucleus using a DAPI filter set and the GFP tagged protein using a GFP FITC 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 2 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 3 Protect samples from light and incubate for 15 to 30 m
10. he 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 2 Dual Detection Reagent The concentration of Mito ID Red 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 10 mL of 1X Assay Buffer see preparation in step 1 or cell culture medium add 1 L of Mito ID Red Detection Reagent and 10 L 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 An intermediate 10 fold dilution of the Mito ID Red Detection Reagent can be made in DMSO if larger staining volumes are not needed The intermediate dilution is stable for at least 4 weeks if stored at 20 C c The Hoechst 33342 Nuclear Stain can be diluted further if its staining intensity is much stronger than t
11. inutes at 37 C 4 Wash the cells with 100 L 1X Assay Buffer Remove excess buffer Resuspend cells in 100 L 1X Assay Buffer then apply the cells to a glass slide and overlay with a coverslip 5 Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard Rhodamine or Texas Red filter set for imaging the mitochondria Optionally image the nucleus using a DAPI filter set and the GFP tagged protein using a GFP FITC filter set 4 D ALDEHYDE FIXATION AND DETERGENT PERMEABILIZATION OF LIVE CELLS STAINED WITH MITO ID RED DYE NOTE It is NOT recommended to stain with both Mito ID Red and the Hoechst nuclear counterstain if they are to be fixed The recommended method of staining fixed cells is to stain after the cells have been fixed 1 Stain the cells as described in section C using a 2 500 fold dilu tion of the Mito ID Red dye instead of the 10 000 fold dilution After staining with Mito ID Red dye wash the cells in fresh pre warmed growth medium or 1X Assay Buffer 2 Carefully remove the growth medium or 1X Assay Buffer covering the cells and replace it with freshly prepared medium or buffer containing 3 7 formaldehyde NOTE If the growth medium contains serum the formaldehyde solution should also be prepared in growth medium containing serum 3 Incubate the cells at 37 C for 15 minutes 4 After fixation wash the cells several times in
12. istance in selecting optimal filter sets for your microscope 6 Figure 1 Absorption and fluorescence emission spectra ex em 558 690nm for Mito ID Red A and Hoechst 33342 ex em 350 461nm 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 once the mitochondrial membrane potential is dissipated Mito ID Red 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 perturbing drugs such as valinomycin In addition to being a live cell permeable dye Mito ID Red dye is also retained during or after cell fixation and detergent permeabilization Mito ID Red dye has been shown to co localize with EGFP cytochrome C oxidase chimeric protein in the HeLa TurboGreen mitochondria cell line HeLa mitoGFP MarinPharm GmbH Lucken walde Germany Typically intense red fluorescent staining of the mitochondrial network i
13. n the perinuclear region of mammalian cells is readily apparent using Mito ID Red dye often with a more grain like structure in the sub plasma membrane region Mito ID Red dye co localizes with the EGFP cytochrome C oxidase signal demonstrating selectivity for mitochondria 7 VIII Troubleshooting Guide Problem Potential Cause Suggestion Mitochondria are not suffi ciently stained Very low concentration of Mito ID Red dye was used or dye was incubated with the cells for an insufficient length of time Either increase the labeling concentration or increase the time allowed for the dye to accumulate in the mito chondria once the cells have been transferred to fresh medium Mito ID Red dye is non specifically binding to the surface We have noted that certain cell culture surfaces with associated gaskets can absorb the dye Grow cells on glass cover slips or try another type of slide Precipitate is seen in the 10X Assay Buffer Precipitate forms at low temperatures 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 red 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 ser
14. 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 claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo CELLestial GFP Certified and Mito ID are trademarks of Enzo Life Sciences Inc Several of Enzo s products and product applications are covered by US and foreign pat ents and patents pending Contents I Introduction 1 II Reagents Provided and Storage 2 III Additional Materials Required 2 IV Safety Warnings and Precautions 2 V Methods and Procedures 3 A REAGENT PREPARATION 3 B STAINING LIVE ADHERENT CELLS 4 C STAINING LIVE CELLS GROWN IN SUSPENSION 4 D ALDEHYDE FIXATION AND DETERGENT PERMEABILIZATION OF LIVE CELLS STAINED WITH MITO ID RED DYE 5 E STAINING OF ALDEHYDE FIXED AND DETERGENT PERMEABILIZED CELLS 6 VI Appendices 6 A FILTER
15. um to stain and wash solutions Serum does not affect staining Normal amounts of serum added range from 2 to 10 8 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 Mitotracker 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 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 usa enzolifesciences 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 664 951
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