51006-500 User Manual
Contents
1. A Enzo D 00000 LU D 000 00000 LU Lu 00000 LU oa Total Nuclear ID Green Red Nucleolar Nuclear Detection Kit Instruction Manual Cat No 51006 500 500 assays For research use only Rev 1 0 February 2009 Notice to Purchaser The Total Nuclear ID Green Red Nucleolar Nuclear Detection Kit 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 imaging applications such as confocal microscopy flow cytometry and HCS where consistency and reproduci bility 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 obli2. 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 recommended 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 Dual Detection Reagent The concentration of Nucleolar ID Green and Nuclear ID Red dyes for optimal staining will vary depending upon the appli cation Suggestions are provided to use as guidelines though some modifications may be required depending upon the particu lar 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 To each mL of 1X Assay Buffer see preparation in step 2 page 3 or cell culture medium containin
3. trafficking and localization arising 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 fluorescently 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 processing 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 s
4. 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 Total Nuclear ID Green Red Nucleolar Nuclear Detection Kit typically display two prominent nucleoli per cell while U2OS 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 induced by actinomycin D is likely to coincide with rRNA degradation events known to occur during treatment with this drug However it is not definitively established 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 Detection Reagent does bind nucleic acids in solution but does not show significant selectivity towards RNA relative to DNA in such n vitro experiments References 1 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 Nucl
5. ZO LIFE SCIENCES GmbH Marie Curie Strasse 8 DE 79539 Lorrach Germany Tel 48 0 7621 5500 526 Fax 490 7621 5500 527 infa de amp venzolifesciences com incorporating UK amp Ireland ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX BNL UK Tel 0845 601 1483 UK customers Tel 44 0 1392 825800 nverseas Fax 444 0 1392 825810 info uk amp enzolifesciences com For Local Distributors please visit our Website LEXIS BIOMOL BIOCHEMICALS
6. ardous 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 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 t is supplied lyophilized 125 ug and should be centrifuged briefly to gather the material at the bottom of
7. ensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm through its disruption The Total Nuclear ID Red Green Nucleolar Nuclear Detection Kit is specifically designed for visualizing nucleoli and nuclei in living cells Both dyes in the kit are resistant to photobleaching facilitating their use in imaging applications Since it is difficult to study nucleolar dynamics using fixed and permeabilized cells a fluorescent nucleolus selective live cell imaging dye becomes very useful in examining changes in this organelle in relation to the organization of the DNA within the cell nucleus as revealed by the supplied DNA counterstain 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 synthe sis inhibitor Nucleolar synthesis of ribosomal RNA is especially sensitive to actinomycin D Potential applications for this kit include monitoring of impaired ribosome biogenesis inhibition of transcription cell cycle dynamics cellular stress distribution trafficking and dynamics of nucleolar proteins distribution 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 Avo
8. eolus the fascinating nuclear body Histochem Cell Biol 129 13 31 3 Smetanaa Buscha Chana Smetana and Busch 2001 Immunocytochemical localization of nucleophosmin and RH Il Gu protein in nucleoli of HeLa cells after treatment with actinomycin D Acta Histochemica 103 3 325 334 Troubleshooting Guide Potential Cause Suggestion Nucleoli are not sufficiently stained Nuclei 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 dye is too bright compared to the red nuclear stain Cells do not appear healthy Actinomycin D Control does not go into solution Actinomycin D 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 Very low concentration of Nuclear ID Red dye was used or dye was incu bated with the cells for an insufficient length of time The Nucleolar ID Green 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 solub
9. g serum add 1 uL of Nucleolar ID Green Detection Reagent and 1 uL of Nuclear ID Red Detection Reagent Serum may be included if preferred NOTE a The dyes may be combined into one staining solution or each may be used separately if desired b The concentration of the Nucleolar ID Green dye may be decreased to equal the staining intensity of the Nuclear ID Hed dye B 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 Dual Detection Reagent see sec tion V A3 above to cover the monolayer cells 100 uL of label ing 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
10. gation 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 Nucleolar ID and Nuclear 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 1 eec ll Reagents Provided and Storage Ill Additional Materials Required IV Safety Warnings and Precautions V Methods and Procedures A Reagent Preparation B Cell Preparations occi C Staining Live Adherent Cells D Staining Live Cells Grown in Suspension VEE tADDENGICGS A Filter Set Selection enm Er TRESS ur dede es E VII References aisi vest veb ue DE Troubleshooting Guide Introduction Enzo Life Sciences Total Nuclear ID Green Red Nucleolar Nuclear Detection Kit contains two proprietary dyes suitable for simultaneous live cell staining of nucleoli and nuclei The dyes allow examination of nucleo lar dynamic changes in intracellular distribution
11. id 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 Nucleolar ID Green Detection Reagent Nuclear ID Red 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 Glass microscope slides Glass cover slips Deionized water Anhydrous DMSO optional Growth medium e g Dulbecco s Modified Eagle Medium D MEM Safety Warnings and Precautions This product is for research use only and is not intended for diagnos tic purposes The Nucleolar ID Green and Nuclear ID Red detection reagents contain 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 han dling these reagents 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 haz
12. ility 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 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 stain or shorten the expo sure time Add serum to the staining solution Serum does not affect staining Normal amounts of serum added range from 296 to 1096 Warm the solution to 37 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 Butler Pike Plymouth Meeting 19462 1202 USA Tel 1 800 942 0430 610 941 0430 Fax 610 841 9252 info usa Penzolifesciances com Switzerland amp Rest of Europe LIFE SCIENCES Industriestrasse 17 Postfach CH 4415 Lausen Switzerland Te 41 061 926 89 89 Fax 41 061 826 89 79 info ch amp enzolifasciances com Benelux ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium Tel 32 0 3 466 04 20 Fax 432 03 466 04 29 info betenzolifesciances com Germany EN
13. slide Analyze the stained cells by wide field fluorescence or confocal 4 microscopy 60X magnification recommended Use a standard FITC filter set for imaging the nucleolus and a standard Rhoda mine or Texas Red filter set for imaging the nucleus D 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 A3 page 4 to cover the dispersed cell pellet Protect samples from light and incubate for 15 to 30 minutes at 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 Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC filter set for imaging the nucleolus and a standard Rhoda mine or Texas Red filter set for imaging the nucleus 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 gt 22 7 m
14. u X Fluarescance emission Absorption Fluorescance omission d SOG S50 400 450 DOU S50 600 650 TOG DO amu 5480 D On GO PONO ove beneath nam eres Lem nem Figure 1 Absorption and fluorescence emission spectra for Nucleolar ID Green A and Nuclear ID Red B dyes All spectra were deter mined in 1X Assay Buffer VII B 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 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 Nuclear ID Red Detection Reagent maximally stains the DNA in the cell nucleus Both dyes display high cellular plasma and nuclear membrane permeability and are well tolerated by living cells The number of nucleoli in different cell types observed with the Nucleolar ID
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