User Manual-ENZ-51005-500 - Rev 1.1.1 April 2011.pub
Contents
1. VIII Troubleshooting Guide Problem Potential Cause Suggestion Acidic organelles are not sufficiently stained Very low concentration of Lyso 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 lyso some once the cells have been transferred to fresh medium The fluorescence of Lyso ID Red dye appears to spread laterally out of the acidic organelles and dissi pate within the cell during prolonged observation by fluorescence microscopy Too intense incident illumi nation is being employed with the dye Other com mercial dyes will rapidly photobleach under similar conditions but Lyso ID Red dye is very photostable Insert neutral density filters into the optical illumination path or reduce duration of exposure to the incident light Lyso ID Red dye fails to stain acidic organelles in fixed and or permeabilized cells The dye is only suitable for live cell staining Use the dye only for live cell analysis 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 lysosomal stain Different microscopes cameras and filters may make some signals appear very bright2. 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 Chloroquine treated cells apper dead or are no longer attached to the surface The EC50 of chloroquine may be different with different cell lines Try lowering the dose of Chloroquine or shortening the time of exposure 7 www enzolifesciences com 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 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 49 0 7621 5500 527 E info de enzolifesciences com www enzolifesciences com UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 0845 601 1488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info uk enzolifesciences com www enzolifesciences com www enzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 41 0 61 926 89 89 F 41 0 61 926 89 79 E info ch enzolifesciences com www enzolifesciences
3. Life Sciences Lyso ID Red Lysosomal Detection Kit GFP Certified contains a novel acidic organelle selective dye suitable for live cell staining Conventional fluorescent stains for acidic organelles such as Acridine Orange Catalog No 52405 form metachromatic artifacts that interfere with multicolor imaging applications Lyso ID Red dye generates emission profiles that can be multiplexed with other fluorophores The dye accumulates in acidic compartments such as endosomes lysosomes and secretory vesicles Low micromolar concentrations of Lyso 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 Lyso ID Red dye is in fluorescence 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 interactions between these molecules For example dual color fluorescence detection may be employed for the identification of vesicular compartments and the investigation of their dynamics and fusion during exocytosis However to date photoconversion of red fluorescent dyes to green has been problematic as observed with Lysotracker Red DND 99 dye In addition metachromatic artifacts wherei
4. Lyso ID Red Lysosomal Detection Kit GFP Certified Instruction Manual Cat No ENZ 51005 500 500 assays For research use only Rev 1 1 1 April 2011 Enabling Discovery in Life Science Notice to Purchaser The Lyso ID Red Lysosomal Detection 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 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 purchas
5. com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 32 0 3 466 04 20 F 32 0 3 466 04 29 E info be enzolifesciences com www enzolifesciences com FRANCE ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert Einstein FR 69100 Villeurbanne France T 33 472 440 655 F 33 437 484 239 E info fr enzolifesciences com www enzolifesciences com Enabling Discovery in Life Science
6. U2OS cells treated with 300 M chloroquine for 4 hours show a dramatic increase in lysosome like vesicle number and volume confirming Lyso ID Red dye is associ ated with this subcellular compartment Thus Lyso ID Red stain can be employed to highlight lysosome like organelles under certain conditions such as chloroquine drug treatment wherein cells produce lysosome like bodies that contain most of the degradative enzymes of the lysosome but are not as acidic as this organelle 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 Jaiswal Fix Takano Nedergaard and Simon 2007 Resolving vesicle fusion from lysis to monitor calcium triggered lysosomal exocytosis in astrocytes Proc Natl Acad Sci U S A 104 35 14151 14156 4 Brunk Dalen Roberg and Hellquist 1997 Photo oxidative disrup tion of lysosomal membranes causes apoptosis of cultured human fibroblasts Free Radical Biology and Medicine 23 4 616 626 5 Michihara Toda Kubo Fujiwara Akasaki and Tsuji Disruptive effect of chloroquine on lysosomes in cultured rat hepatocytes 2005 Biol Pharm Bull 28 6 947 951 6
7. clear counterstain is also provided in the kit 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 IV Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes The Lyso 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 eye
8. e 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 Lyso ID are trademarks of Enzo Life Sciences Inc Lysotracker is a trademark of Molecular Probes Several of Enzo s products and product applications are covered by US and foreign patents 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 Cell Preparations 3 C Staining Live Adherent Cells 4 D Staining Live Cells Grown in Suspension 5 VI Appendices 5 A Filter Set Selection 5 B Results 6 VII References 6 VIII Troubleshooting Guide 7 I Introduction Enzo
9. lows To each mL of 1X Assay Buffer see preparation in step 2 or cell culture medium add 1 L of Lyso ID Red Detection Reagent and 1 L of Hoechst 33342 Nuclear Stain 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 Hoechst 33342 Nuclear Stain can be diluted further if its staining intensity is much stronger than the red lysosomal stain Lyso 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 CELL PREPARATIONS Cells should be maintained via standard tissue culture practices Positive control cells should be pretreated with the chloroquine con trol for 2 8 hours Response to chloroquine 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 C 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 A3 page 3 to cover the monolayer cell
10. n fluorescent dyes emit both in the red and green regions of the spectrum are evident as observed with Acridine Orange dye These observations 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 associate with the target organelle requiring imaging within a minute or two of dye addition 3 4 Enzo s Lyso ID Red dye a new red emitting cell permeable small organic probe molecule that spontaneously localizes to live cell acidic organelles was developed to overcome the above problems The Lyso ID Red dye can be readily used in combination with other common UV and visible light excitable fluorescent dyes and various fluorescent proteins in multicolor imaging and detection applications The dye is suitable for both short term and long term tracking studies Lyso ID Red dye emits in the Texas Red region of the visible light spectrum and is highly resistant to photobleaching concentration quenching and photoconversion The Lyso ID Red Lysosomal Detection Kit GFP Certified is specifically designed for use with GFP expressing cell lines as well as cells express ing blue cyan or yellow fluorescent proteins BFPs CFPs YFPs A lysosome perturbation agent chloroquine is provided as a positive control for monitoring changes in lysosome number and volume A nu
11. osomes The chloroquine provided in the kit may be used as a positive control for increasing lysosome number and volume It is supplied lyophilized 7 5 moles and should be centrifuged briefly to gather the material at the bottom of the tube Reconstitute the lyophilized material in 125 L deionized water for a 60 mM stock solution It is recommended that treatment with the agent be performed using 10 300 M final concentration in order to observe changes in lysosomal morphology Unused stock chloroquine may be stored in small aliquots at 20 C for several weeks 2 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 Lyso 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 3 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 fol
12. s 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 lysosomes Optionally image the nucleus using a DAPI filter set and the GFP tagged protein using a GFP FITC filter set 4 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 2 Carefully remove the supernatant by aspiration and dispense sufficient volume of Dual Detection Reagent see section V A3 page 3 to cover the dispersed cell pellet 3 Protect samples from light and incubate for 15 to 30 minutes 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 lysosomes Optionally image the nucleus using a DAPI filter set and the GFP tagged protein using a GFP FITC filter set VI APPENDICES A Filter Set Selec
13. tion 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 Figure 1 Absorption and fluorescence emission spectra for Lyso ID Red A and Hoechst 33342 B dyes All spectra were determined in 1X Assay Buffer 5 B Results Lysosomes are membrane bound organelles involved in the degrada tion of macromolecules and pathogens in diverse processes including endocytosis phagocytosis and autophagy Lysosomal morphology varies with the state of the cell and its degree of degradative activity and the vesicles can have pieces of membranes vacuoles granules and even parts of mitochondia within them They are typically spherical vesicles ranging in size from 0 2 to 2 m in diameter The lumen of lysosomes and other acidic organelles is characterized by low pH generated via proton pumping vacuolar ATPases Lyso ID Red dye is selectively sequestered in acidic organelles by a mecha nism that likely involves protonation and retention within the membranes of the organelles However staining is even feasible in cells pretreated with weakly basic cell permeant compounds such as chloroquine and the fluorescence emission of the dye itself is largely independent of pH HeLa and
14. wear 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 Reagent Quantity Lyso ID Red Detection Reagent 50 L Hoechst 33342 Nuclear Stain 50 L Chloroquine Control 7 5 mol 10X Assay Buffer 15 mL 2 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 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 Positive Control Chloroquine is a lysosomotropic agent It accumulates preferen tially in the lysosomes of cells 5 The pKa for the quinoline nitrogen of chloroquine is approximately 8 5 At physiological pH it is roughly 10 deprotonated as calculated using the Henderson Hasselbalch equation This decreases to roughly 0 2 at a lysosomal pH of 4 6 Since the deprotonated form of the compound is more membrane permeable than the protonated form chloroquine becomes quantitatively trapped in lys
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