User Manual-ENZ-51028-K100
Contents
1. preferably with disposable plas tic tips e Adjustable speed centrifuge with swinging buckets for suspension cul tures 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 protocol e PBS optional for fixation protocol Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes Some components of this kit may contain hazardous substances Reagents be harmful if ingested or absorbed through the skin and may cause irritation to the eyes They should be treated as possible mutagens 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 To avoid photobleaching perform all manipulations in low light environments or protected from light by other means Methods and Procedures NOTE PLEASE READ THE ENTIRE PROCEDURE BEFORE STARTING Allow all reagents to thaw at room temperature before starting with the pro cedures Upon thawing gently hand mix or vortex the reagent2. region of the visible light spectrum and is resistant to photo bleaching concentration quenching photo conversion The kit should also be suitable for identifying Golgi body perturbing agents and thus can be a useful tool for examining retrograde flow mechanisms in cellular secretory pathways The Golgi ID Green Assay Kit has been specifically designed for use with RFP expressing cell lines as well as cells expressing blue cyan or orange fluorescent proteins BFPs CFPs OFPs Additionally the kit is suitable for use with live or fixed cells in conjunction with probes such as labeled antibodies or other fluorescent conjugates displaying similar spectral properties as Texas Red or coumarin A blue nuclear counter stain 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 upright and protected from light at lt 20 C 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 100 microscopy assays using live cells adherent or in suspension Reagent Quantity Golgi IDTM Green Detection Reagent 50 nmoles Hoechst 33342 Nuclear Stain 50 uL 10X Assay Buffer 1 15 mL 50X Assay Buffer 2 1 2 mL Additional Materials Required e Standard fluorescence microscope e Calibrated adjustable precision pipets
3. DAE AAA DD DD oo x unn LETIEIEI unn n n Enzo Enabling Discovery in Life Science Golgi ID Green Assay Kit for detection of Golgi bodies by microscopy Instruction Manual Cat No ENZ 51028 K100 for 100 assays For research use only Rev 1 0 March 2010 Notice to Purchaser The Golgi ID Green Assay Kit is a member of the CELLestial product line reagents 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 This product is offered under a limited warranty The product is guaranteed to meet appropriate specifications described i
4. NG ALDEHYDE FIXED CELLS 1 Grow cells on coverslips inside a Petri dish filled with the appropri ate culture medium When the cells have reached the desired level of confluence carefully remove the medium 2 Wash the cells with 100 uL 1X Assay Solution 3 Fix the cells for 10 minutes at 37 C with 496 formaldehyde solution see section V A4 page 4 Wash the cells 3 times using 100 pL ice cold 1X Assay Solution for each wash Dispense sufficient volume of Dual Detection Reagent see sec tion V A3 page 4 to cover the monolayer cells 100 pL for cells grown on an 18 X 18 mm coverslip Protect samples from light and incubate for 30 minutes at 4 C 7 Wash the cells times using 100 uL 1X Assay Solution for each 10 wash Add 100 uL fresh medium to cover the cells and allow to incubate at room temperature for 45 minutes 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 Golgi bodies Optionally image the nucleus using a DAPI filter set and the RFP tagged protein using a Texas Red filter set NOTE Avoid the use of detergents and ethanol methanol acetone fixatives VI APPENDICES A FILTER SET SELECTION The selection of optimal filter sets for a fluorescence microscopy application requires m
5. atching 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 300 350 400 450 500 550 600 650 700 200 250 300 350 400 450 500 550 600 Wavelength nm Wavelength nm Figure 1 Fluorescence excitation and emission spectra for Golgi ID Green dye panel A and absorbance and fluorescent emission spectra for Hoechst 33342 dye panel B All spectra were determined in 1X Assay Solution B RESULTS The Golgi apparatus is an organelle found in eukaryotic cells respon sible for processing and packaging proteins and lipids during their subcellular sorting One prominent post translational modification proteins and lipids undergo within the Golgi apparatus is glycosylation through the actions of various glycosidases and transferases Interfering with Golgi apparatus function by for example treatment with certain drugs typically leads to perturbation of Golgi function including blocking of secretion altering morphology dispersion of Golgi structural elements loss of cisternal stacks or inhibition of vesicular transport The Golgi ID Green Assay Kit provides rapid and convenient method for visualizing the Golgi body within living cells without a requir
6. ement for lengthy transfection procedures In addition to serv ing as a live cell permeable dye Golgi ID Green dye is also local ized within the Golgi body of fixed cells Upon staining with the dual detection reagent the nucleus should fluoresce blue as detected with a DAPI filter set and the Golgi apparatus should exhibit prominent green fluorescence appearing as a perinuclear reticular network within the cell when employing a FITC filter set Only minor staining of other membranes within the cell should be observed Vil References 1 Deng Y Bennink JR Kang HC Haugland RP Yewdell JW Fluores cent conjugates of brefeldin A selectively stain the endoplasmic reticulum and Golgi complex of living cells J Histochem Cytochem 1995 Sep 43 9 907 15 Dinter A and Berger EG Golgi disturbing agents Histochem Cell Biol 1998 May Jun 109 5 6 57 1 90 VIII Troubleshooting Guide Problem Potential Cause Suggestion Golgi apparatus is not suffi ciently stained Very low concentration of Golgi IDTM Green dye was used or cells were incubated too long after labeling Either increase the labeling concentration or limit the time allowed for the cells to grow after the dye has been removed We recommend labeling for 30 minutes and incubating cells for 30 min utes after the label has been removed Precipitate is seen in the 10X Assay Buffer 1 Precipitate forms at low temperatures Allow solution to
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8. excess buffer and place coverslip on slide 8 Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC filter set for imaging the Golgi bodies 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 then wash the cells with 200 uL of 1X Assay Solution from section V A1 page 3 Carefully remove the supernatant by aspiration and then add 100 uL of Dual Detection Reagent see section V A3 page 4 to the cell pellet 4 Protect samples from light and incubate for 30 minutes on ice 5 Wash the cells 2 times using 200 pL ice cold medium for each wash Re suspend the cells in 100 ice cold medium and then incu bate the cells at 37 C for 30 minutes 7 Wash the cells with 200 uL 1X Assay Solution 8 Re suspend cells in 100 uL 1X Assay Solution then transfer 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 Golgi bodies Optionally image the nucleus using a DAPI filter set and the RFP tagged protein using a Texas Red filter set D STAINI
9. ll staining Compared with other commercially available dyes for labeling Golgi bodies Golgi ID Green dye is more faithfully localized to the target organelle with minimal staining of the endoplasmic reticulum lar concentrations of Golgi ID Green dye are sufficient for staining mam malian cells as validated with human cervical carcinoma cell line HeLa human T lymphocyte cell line Jurkat canine kidney cell line MDCK and human bone osteosarcoma epithelial cell line U2OS One important application of Golgi 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 their associations and interactions Many organelle targeting probes photo bleach rapidly are subject to quenching upon concentration in organelles are highly toxic or only transiently associate with the target organelle requiring imaging within a minute or two of dye addition Consequently Golgi ID Green dye a new green emitting cell permeable small molecule organic probe that spontaneously localizes to live or fixed Golgi apparatus was developed Golgi ID Green dye can be readily used in combination with other common UV and visible light excitable organic fluorescent dyes and various fluorescent proteins in multicolor imaging and detection applica tions The dye emits in the FITC
10. n 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 Golgi 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 l TREO GU CUI ON ness eii ll Reagents Provided and Storage Additional Materials Required IV Safety Warnings and Precautions V Methods and Procedures A REAGENT PREPARATION aa B STAINING LIVE ADHERENT CELLS C STAINING LIVE CELLS GROWN IN SUSPENSION D STAINING ALDEHYDE FIXED CELLS VI Appendices A FILTER SET SELECTION seen B RESULTS Ie u g sa Areva and Sa VII References VIII Troubleshooting Guide Introduction Enzo Life Sciences Golgi ID Green Assay Kit contains a Golgi apparatus selective dye suitable for live cell or aldehyde fixed ce
11. s 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 Solution Allow the 10X Assay Buffer 1 the 50X Assay Buffer 2 to warm to room temperature Make sure that the reagents are free of any crystallization before use Prepare enough 1X Assay Solution for the number of samples to be assayed For each 10 mL preparation of 1X Assay Solution add 1 mL 10X Assay Buffer 1 and 0 2 mL 50X Assay Buffer 2 into 8 8 mL deionized water Mix well 2 100X Golgi ID Green Dye Solution Add 100 pL of the 1X Assay Solution prepared above to the vial containing lyophilized Golgi ID Green Detection Re agent 50 nmoles Vortex gently or slowly rotate the tube to dissolve 3 Dual Detection Reagent The concentration of Golgi ID TM 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 milliliter of 1X Assay Sol
12. ution see preparation in section V A1 add 10 uL of the 100X Golgi ID Green Dye Solution from section V A2 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 Golgi stain 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 496 Formaldehyde Solution The following procedure is for preparation of 10 mL of 496 formadehyde solution Dilute 0 4 gram paraformaldehyde to a final volume of 10 mL with PBS Mix well B STAINING LIVE ADHERENT CELLS 1 Grow cells on coverslips inside a Petri dish filled with the appropri ate culture medium When the cells have reached the desired level of confluence carefully remove the medium Wash the cells with 100 1X Assay Solution from section V A1 page 3 Dispense sufficient volume of Dual Detection Reagent see section V A3 above to cover the monolayer cells 100 uL for cells grown on an 18 X 18 mm coverslip 4 Protect samples from light and incubate for 30 minutes at 4 C 5 Wash the cells 3 times using 100 uL ice cold medium for each wash 6 Incubate the cells in medium at 37 C for 30 minutes 7 Wash the cells with 100 uL 1X Assay Solution Remove
13. warm to room temperature or 37 C then vortex to dissolve all precipitate The blue nuclear counter stain is too bright compared to the green Golgi 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 The green Golgi dye ap pears to stain more than just the Golgi bodies Excess Golgi ID Green dye was used or cells were not washed well enough after staining with medium containing serum Reduce the concentration of Golgi ID green and or add additional washes with ice cold medium containing 10 serum and extend the incubation time in medium after staining c 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 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 441 061 926 89 89 F 441 061 926 89 79 E info 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 enzolifesci
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