User Manual-ENZ-51026-K500
Contents
1. 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 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 2 3 7 Formaldehyde Solution The following procedure is for preparation of 10 mL of 3 7 formaldehyde solution Dilute 0 37 gram paraformaldehyde to a final volume of 10 mL with 1X Assay Buffer Mix well 3 0 1 Triton X 100 optional The following procedure is for preparation of 10 mL of 1 Triton X 100 solution Dilute 10 uL Triton X 100 to a final volume of 10 mL with 1X Assay Buffer or 1X Assay Buffer containing 2 serum Mix well 4 Dual Detection Reagent The concentration of ER 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 overlo2. 1 Wash the cells in 1x Assay Buffer from step A 1 page 3 2 Carefully remove the buffer covering the cells and replace it with freshly prepared 3 7 formaldehyde solution 3 Incubate the cells at 37 C for 10 minutes 4 After fixation rinse the cells several times in 1X Assay Buffer 5 Optional If cells are to be labeled with an antibody permeabiliza tion step is recommended to enhance the antigen s accessibility This is done by incubating the fixed cells in 0 1 Triton X 100 from step A 3 page 3 at room temperature for 1 minute 6 Following permeabilization rinse the cells with 1X Assay Buffer 7 Perform staining as recommended for adherent or suspension cells see sections B or C NOTE When performing standard immunofluorescence staining proto cols using a fluorescein or coumarin labeled antibodies or equivalent administer post fixation according to manufacturer instructions E ALDEHYDE FIXATION AND DETERGENT PERMEABILIZATION OF STAINED LIVE CELLS Live cells stained with ER ID Red dye may be fixed with formalde hyde and permeabilized with Triton X 100 Fixation and permeabili zation makes it possible to probe for other intracellular structures by conventional immunofluorescence labeling methods The ER ID Red dye is retained following fixation and permeabilization using the protocol described below 1 Wash the ER ID Red stained cells with 1x Assay Buffer from step A 1 page 3 2 Carefu
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4. JAE NAAR AL w 7 Enzo Enabling Discovery in Life Science og og CIEE fe i m ooog oog o0oo00 m mm f aae G am a a a Eee mme ff ome am odd odd odd o0 x ooo ER ID Red Assay Kit GFP Certified for detection of endoplasmic reticulum by microscopy Instruction Manual Cat No ENZ 51026 K500 500 assays For research use only Rev 1 0 February 2010 Notice to Purchaser The ER ID Red Assay Kit GFP Certified 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 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 g
5. Troubleshooting Guide sssssnsennennnennnnnnnnnnnnnnnnnnne Introduction Enzo Life Sciences ER ID Red Assay Kit GFP Certified contains a novel endoplasmic reticulum selective dye suitable for live cell or deter gent permeabilized aldehyde fixed cell staining Micromolar concentrations of ER ID Red dye are sufficient for staining mammalian cells as vali dated with human cervical carcinoma cell line human T lymphocyte cell line Jurkat HeLa and human bone osteosarcoma epithelial cell line U20S One important application of ER ID Red dye is in fluorescence co localization imaging with green fluorescent protein GFP tagged proteins a powerful approach for determining the targeting of molecules to intracel lular compartments and for screening of their associations and interactions However to date photoconversion of red fluorescent dyes to green fluo rescent ones 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 Additionally many organelle targeting probes photobleach rapidly are subject to quenching upon con centration in organelles are highly toxic or only transiently associate with the target organelle requiring imaging within a minute or two of dye addi tion Consequently ER ID Red dye a new red emitting cell permeable small molecule organic probe that spontaneously locali
6. ading 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 milliliter of 1X Assay Buffer see preparation in step V A1 or 1X Assay Buffer containing 2 serum add 1 uL of ER ID Red 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 red endoplasmic reticulum stain ER ID Red 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 or tissue culture treated slides inside a Petri dish filled with the appropriate 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 Wash the cells with 100 uL 1X Assay Buffer Remove excess buffer and place coverslip on slide Analyze the stained cells by wide field fluo
7. cellular organelles found in eukaryotic cells responsible for sorting most of the proteins and lipids of the cell In addition to being a live cell permeable dye ER ID Red dye is also partially retained during or after cell fixation and detergent per meabilization ER ID Red dye has been shown to co localize with EGFP calreticulin chimeric protein in a transduced HeLa cell line Typically intense red fluorescent staining of the endoplasmic reticulum in the perinuclear region of mammalian cells is readily apparent using ER ID Red dye The ER ID Red dye co localizes with the EGFP calreticulin signal demonstrating selectivity for endoplasmic reticula Vil References 1 Freundt Czapiga and Lenardo 2007 Photoconversion of Lyso tracker Red to a green fluorescent molecule Cell Res 17 11 956 958 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 Minamikawa Sriratana Williams Bowser Hill and Nagley 1999 Chloromethyl X rosamine MitoTracker Red photosensitises mito chondria and induces apoptosis in intact human cells Journal of Cell Science 112 2419 2430 Scorrano Petronilli Colonna Di Lisa and Bernardi 1999 Chloro methyltetramethylrosamine Mitotracker Orange Induces the Mito chondrial Permeability Transition and Inhibi
8. dard fluorescence microscope e Calibrated adjustable precision pipetters preferably with disposable plastic tips e Adjustable speed centrifuge with swinging buckets for suspension cultures e Glass microscope slides e Glass cover slips e Deionized water e Anhydrous DMSO optional e Growth medium e g Dulbecco s Modified Eagle Medium D MEM e Paraformaldehyde optional for fixation e Triton X 100 optional for permeabilization Safety Warnings and Precautions e This product is for research use only and is not intended for diagnostic purposes e The ER 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 appro priate precautions when handling e Reagents should be treated as possible mutagens and should be handled with care and disposed of properly e 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
9. lly remove the buffer covering the cells and replace it with freshly prepared 3 7 formaldehyde solution 3 Incubate the cells at 37 C for 10 minutes 4 After fixation rinse the cells several times in 1X Assay Buffer 5 Optional If the cells are to be labeled with an antibody perme abilization step is recommended to enhance the antigen s acces sibility This is done by incubating the fixed cells in 0 1 Triton X 100 from step A 3 page 3 at room temperature for 1 minute Note The staining will not be as intense after fixation of cells It is recommended to fix prior to staining cells Section D 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 Fluorescence Excitation Fluorescence Emission Absorbance Fluorescence Emission 400 450 500 550 600 650 700 750 200 250 300 350 400 450 500 550 600 Wavelength nm Wavelength nm Figure 1 Fluorescence excitation and emission spectra for ER ID Red dye panel A and absorbance and fluorescent emission spectra for Hoechst 33342 dye panel B All spectra were determined in 1X Assay Buffer RESULTS Endoplasmic reticula are sub
10. rescence or confocal microscopy 60X magnification recommended Use a standard Rhodamine or Texas Red filter set for imaging the endoplasmic reticulum 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 f 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 the cells with 100 uL 1X Assay Buffer Remove excess buffer Resuspend cells in 100 uL 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 endoplasmic reticulum Optionally image the nucleus using a DAPI filter set and the GFP tagged protein using a GFP FITC filter set D STAINING OF ALDEHYDE FIXED AND DETERGENT PERMEABILIZED Live CELLS WITH ER ID Rep DYE The ER ID Red dye is capable of staining fixed and permeabilized cells Fixation and permeabilization makes it possible to probe for other intracellular structures by conventional immunofluorescence labeling methods
11. ts Respiratory Complex Implications for the mechanism of cytochrome c release J Biol Chem 274 35 24657 24663 VIII Troubleshooting Guide Problem Potential Cause Suggestion Endoplasmic reticula are not sufficiently stained Very low concentration of ER 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 endo plasmic reticula 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 endoplasmic reticulum 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 10 www enzolifesciences com Enabling Discovery in Life Science Enzo Life Sciences North South America ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA Tel 1 800 942 0430 610 941 0430 Fax 610 941 9
12. uaranteed 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 ER 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 l TREO GU CUI ON ness ecdee ieee tcesecte ete tee ences eaticnedeteeseaeteveseptvenne ll Reagents Provided and Storage cssssesseeese lll Additional Materials Required cs ssseeseeeeen IV Safety Warnings and Precautions 0ccseeee V Methods and Procedures ccsccsssssseseeeseesseseenees A REAGENT PREPARATION csscccceseeeseeseeecesecsssaeeeseeuseeseeaees B STAINING LIVE ADHERENT CELLS 0cc cccceeeeeeeeeeeeeeeeeee C STAINING LIVE CELLS GROWN IN SUSPENSION 00ceeeee D STAINING OF ALDEHYDE FIXED AND DETERGENT PERMEABILIZED LIVE CELLS WITH ER ID RED DYE E ALDEHYDE FIXATION AND DETERGENT PERMEABILIZATION OF STAINED LIVE CELLS 0 0 008 Vi Appendices siie a ennie apa aa aaan A FILTER SET SELECTION inarun teina Be E E i a AEE EASE EE A T SE E VIR Refer NCeS eais cae ssccccccsasdvet secasecsadeseassauceavets caadeaceccuas VIII
13. zes to live or fixed endoplasmic reticula was developed ER ID Red dye can be readily used in combination with other common UV and visible light excitable organic fluorescent dyes and various fluorescent proteins in multi color imaging and detection applications The dye emits in the Texas Red region of the visible light spectrum and is highly resistant to photo bleaching concentration quenching and photoconversion The ER ID Red Assay Kit GFP Certified is specifically designed for use with GFP expressing cell lines as well as 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 Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored upright 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 ER ID Red Detection Reagent Hoechst 33342 Nuclear Stain 10X Assay Buffer Additional Materials Required e Stan
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