User Manual-ENZ-51009-500
Contents
1. 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 Positive Control The antibiotic actinomycin D is a DNA dependent RNA synthesis inhibitor Nucleolar synthesis of ribosomal RNA is especially sensitive to actinomycin D The rearrangement of the nucleolus due to actinomycin D treatment is widely used to examine the localization of various nucleolar components including nucleolar proteins Typically at higher doses of the drug 4 10 ug ml for 4 hours the nucleolus in mammalian cells disappears or where still present is dramatically reduced in amount while at lower concentrations 1 4 ug ml for 4 hours less dramatic reduction in nucleolar amount is often observed The actinomycin D provided in the kit may be used as a positive control for reducing nucleoli size and number is supplied lyophilized 125 ug and should be centrifuged briefly to gather the material at the bottom of the tube Reconstitute the lyophilized material in 250 uL deionized water for a 0 5 mg mL stock solution Vortex vigorously allow to sit for 15 minutes then vortex again to completely bring it into solution It is recommende2. Pike Plymouth Meeting 19462 1202 USA Tel 1 800 947 0430 610 941 0430 Fax 610 941 9252 info usatenzolitesciances com Switzerland amp Rest of Europe LIFE SCIENCES Industriestrasse 17 Postfach CH 4415 Lausen Switzerland Tal 41 061 926 89 89 Fax 41 061 926 89 79 info chPenzolifesciances com Benelux ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium Tal 32 0 3 466 H 20 Fax 432 03 466 04 29 info betenzolifesciances com Germany 20 LIFE SCIENCES GmbH Mane Curie Strasse 8 0 79539 L rrach Germany Tel 49 0 7621 5500 526 400 7621 5500 527 info ceenzolifesciences com incorporating UK amp Ireland ENZO LIFE SCIENCES LTD Palatine House Matford Court Exeter EX UK Tel 0845 601 1483 UK customers Tel 44 0 1392 825000 joverseas Fax 440 1392 825010 info UkKG enzolifesciences com For Local Distributors please visit our Website LEXIS BIOMOL BIOCHEMICALS
3. Reagent at this time CELL PREPARATIONS Cells should be maintained via standard tissue culture practices Positive control cells should be pretreated with the actinomycin D control for 2 6 hours Response to actinomycin D is time and concentration dependent and may also vary significantly depending upon cell type and cell line Negative control cells should be treated with a vehicle DMSO media or other solvent used to reconstitute or dilute an inducer or inhibitor for an equal length of time under similar conditions 1 STAINING LIVE ADHERENT CELLS 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 Detection Reagent see section V A3 above 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 fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC filter set for imaging the nucleolus STAINING LIVE CELLS GROWN IN SUSPENSION 1 2 3 4 5 Centrifuge cells for 5 minutes at 400 x g at room temperature RT to obtain a cell pellet Carefully remove the superna
4. J Enzo Nucleolar ID Green Detection Kit for Fluorescence Microscopy Instruction Manual Cat No 51009 500 500 assays For research use only Rev 1 0 April 2009 Notice to Purchaser The Nucleolar ID Green Detection 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 imaging applications such as confocal microscopy flow cytometry and HCS 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 claims must be made to Enzo Life Sciences Inc within five 5 day
5. d that treatment with the agent be performed using 1 5 ug mL final concentration in order to observe changes in nucleolar morphology Unused stock actinomycin D may be stored in small aliquots at 20 C for several weeks 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 3 Detection Reagent The concentration of Nucleolar ID Green dye for optimal stain ing 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 the Detection Reagent for the number of samples to be assayed as follows To each mL of 1X Assay Buffer See preparation in step 2 page 3 or cell culture medium containing serum add 1 uL of Nucleolar ID Green Detection Reagent Serum may be included if preferred NOTE If desired other stains such as Hoechst 33342 Draq5 or Enzo s Nuclear ID Red dye may be added to the diluted Nucleolar ID Green Detection
6. ed by actinomycin D is likely to coincide with rRNA degradation events known to occur during treatment with this drug However it is not definitively estab lished whether the dye interacts with rRNA with arginine lysine rich sequences in nucleolar proteins or with some other structural feature of nucleoli Nucleolar ID Green dye does bind nucleic acids solution but does not show significant selectivity towards RNA relative to DNA in such in vitro experiments References Hernandez Verdun and Roussel 2003 Regulators of nucleolar functions Progress in Cell Cycle Research Vol 5 Meijer J z quel and Roberge eds Chapter 31 pp 301 308 Sirri Urcuqui Inchima Roussel and Hernandez Verdun 2008 Nucleolus the fascinating nuclear body Histochem Cell Biol 129 13 31 Smetanaa Buscha Chana Smetana and Busch 2001 Immunocytochemical localization of nucleophosmin and RH II Gu protein in nucleoli of HeLa cells after treatment with actinomycin D Acta Histochemica 103 3 325 334 VIII Troubleshooting Guide Potential Cause Suggestion Nucleoli are not sufficiently stained Nucleolar ID Green dye fails to stain the nucleoli in fixed and or permeabilized cells Precipitate is seen in the 10X Assay Buffer The Nucleolar ID Green stain is too bright compared to the red nuclear stain Cells do not appear healthy Actinomycin D Control does not go into solution Actinomycin D
7. from the previous cell cycle Ribosomal DNA rDNA transcription is maximal in the S and phases silent in mitosis and slowly recovers in the phase of the cell cycle The nucleolus is a prominent nuclear structure in cycling cells but of limited size in the terminal stages of cell differen tiation Cells stained with Nucleolar ID Green Detection Reagent show maximal fluorescence signal within the nucleoli and faint fluores cence throughout the nucleus Weak fluorescence is also observed throughout the cytoplasm predominantly associated with mitochon dria The dye displays high cellular plasma and nuclear membrane permeability and is well tolerated by living cells The number of nucleoli in different cell types observed with the Nucleolar ID Green Detection Reagent will vary and they will be of different sizes as well There appears to be an inverse relationship between size and number of nucleoli in mammalian cells For example HeLa human cervical carcinoma cells stained using the Nuclear ID Green Detection Reagent typically display two prominent nucleoli per cell while U2ZOS human bone osteosarcoma epithelial cells are observed to contain a half dozen smaller nucleoli Ribosomal DNA rDNA transcriptional arrest induced by low doses of actinomycin D results in loss of nucleolar staining in both cell lines as observed with the Nucleolar ID Green Detection Reagent in the kit The dissipation of nucleolar signal induc
8. ng from biological processes such as the cell cycle and ribosome biogenesis Historically nucleolus imaging approaches have required much more laborious and time consuming methods such as fluo rescently labeled RNA microinjection fluorescence in situ hybridization FISH or use of fluorescent protein tagged RNA binding proteins GFP or YFP constructs The nucleolus represents a highly dynamic nuclear domain arising from an equilibrium between the level of ribosomal RNA synthesis and the efficiency of ribosomal RNA 2 Although the nucleolus is primarily associated with ribosome biogenesis several lines of evidence demonstrate that it has additional functions such as regulation of mitosis cell cycle progression and proliferation and many forms of stress response and biogenesis of multiple ribonucleoprotein particles Ribosome biogene sis is regulated throughout interphase and ceases during mitosis Thus there is a direct relationship between cell growth and nucleolar activities Nucleoli are well Known to be dramatically modified in cancer cells Additionally a large number of key proteins from both DNA and RNA containing viruses are localized in the nucleolus including the HIV 1 human immunodeficiency virus type 1 Rev and Tat proteins Targeting of viral proteins to the nucleolus not only facilitates virus replication but may also be required for pathogenic processes The nucleolus is also a sensor of stress due to
9. s of receipt of order Trademarks and Patents Enzo CELLestial Nucleolar ID and Nuclear ID are trademarks of Enzo Life Sciences Inc Vybrant and DyeCycle are trademarks of Life Technologies Draq is a trademark of Biostatus Limited Several of Enzo s products and product applications are covered by US and foreign patents and patents pending Contents VII VIII MO dUCHON iiaa a Reagents Provided Additional Materials Safety Warnings and Methods and A REAGENT PREPARATION CELL PREPARATIONS STAINING LIVE ADHERENT D STAINING LIVE CELLS GROWN IN SUSPENSION APPENdICES fu PIETER SET SELECTION RESULT Troubleshooting Guide Introduction Enzo Life Sciences Nucleolar ID Green Detection Kit contains a proprie tary dye suitable for live cell staining of nucleoli The dye allows examina tion of nucleolar dynamic changes in intracellular distribution trafficking and localization arisi
10. tant by aspiration and dispense sufficient volume of Detection Reagent see section V A3 page 4 to cover the dispersed cell pellet Protect samples from light and incubate for 15 to 30 minutes at 37 C Wash the cells with 100 uL 1X Assay Buffer Remove excess buffer Resuspend cells 100 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 nucleolus 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 Absorbance a T m 2 Figure 1 Absorption fluorescence emission spectra for Nucleolar ID Green dye Spectrum was 200 350 400 450 500 550 600 650 700 determined in 1X Assay Wavelength mm Buffer RESULTS Ribosomal RNAs rRNAs are synthesized processed and assem bled with ribosomal proteins in the nucleolus In mammalian cells the nucleolus is disorganized in prophase and reassembled at the end of mitosis using the nucleolar machineries
11. the redistribution of the ribosomal proteins in the nucleoplasm through its disruption The Nucleolar ID Red Detection Kit is specifically designed for visualiz ing nucleoli in living cells by fluorescence microscopy The dye in the kit is resistant to photobleaching facilitating its use in imaging applications This nucleolar stain is compatible with common live cell nuclear stains such as Hoechst 33342 Draq5 Vybrant DyeCycle Ruby Enzo s Nuclear ID Red dye The kit includes a control nucleolus perturbation agent actinomycin D for monitoring changes in nucleolar dynamics Actinomycin D an antibiotic is a DNA dependent RNA synthesis inhibitor Nucleolar synthesis of ribosomal RNA is especially sensitive to actinomycin D Potential applications for this kit include monitoring of impaired ribo some biogenesis inhibition of transcription cell cycle dynamics cellular stress distribution trafficking and dynamics of nucleolar proteins distribu tion of viral proteins and potentially as an aid to identify cancer cells Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored at lt 20 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 Quantit
12. treated cells apper dead or are no longer attached to the surface Very low concentration of Nucleolar ID Green dye was used or dye was incubated with the cells for an insufficient length of time The Nucleolar ID Green Detection Reagent provided in the kit is only suitable for live cell staining Precipitate forms at low temperatures Different microscopes cam eras and filters may make some signals appear very bright Some cells require serum to remain healthy The recommended volume to dissolve actinomycin D is near the limit of solubility Ambient temperature may affect solubility The of actinomycin D may be different with differ ent cell lines Either increase the labeling concentration or increase the time allowed for the dye to accumulate in the nucleoli Use the dye only for live cell analysis Allow solution to warm to room temperature or 37 C then vortex to dissolve all precipitate Reduce the concentration of the Nucleolar ID Green dye or shorten the expo sure time Add serum to the staining solution Serum does not affect staining Normal amounts of serum added range from 2 to 10 Warm the solution to 37 C to dissolve or dissolve in a larger volume Lower the dose of actino mycin D or shorten the time of exposure Enzo Life Sciences www enzolifesciences com North South America ENZO LIFE SCIENCES INTERNATIONAL ING 5120
13. y Nucleolar ID Green Detection Reagent Actinomycin D Control 10X Assay Buffer Additional Materials Required Standard fluorescence microscope Calibrated adjustable precision pipetters preferably with disposable plastic tips Adjustable speed centrifuge with swinging buckets for suspension cultures e Glass microscope slides e Glass cover slips Deionized water Anhydrous DMSO optional Growth medium e g Dulbecco s Modified Eagle Medium D MEM Safety Warnings and Precautions e This product is for research use only and is not intended for diagnos tic purposes e The Nucleolar ID Green Red Detection Reagent contains DMSO which is readily absorbed through the skin DMSO is harmful if ingested or absorbed through the skin and may cause irritation to the eyes Observe appropriate precautions when handling these reagents 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
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