User Manual-ENZ-51034-K500 - Lyso
Contents
1. to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo CELLestial 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 eii ll Reagents Provided and Storage Additional Materials Required IV Safety Warnings and Precautions V Methods and Procedures eene A Reagent Preparation B Cell C Staining Live Adherent D Staining Live Cells Grown in Suspension VI Appendices A Filter Set Selection o RESUS re MIL References u VIII Troubleshooting Guide Introduction Enzo Life Sciences Lyso ID Green Detection Kit contains a novel acidic organelle selective dye suitable for live cell staining Conventional fluores cent stains for acidic organelles such as Acridine Orange Catalog No ENZ 52405 form metachromatic artifacts that interfere with multicolor imaging applica2. 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 Green 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 follows To each milliliter of 1X Assay Buffer see preparation in step 2 or cell culture medium add 1 uL of Lyso ID Green Detection Reagent and 1 uL 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 green lysosomal stain Lyso ID Green c When staining BFP or CFP expressing cells the Hoechst 33342 Nuclear Stain should be omitted due to its spect
3. J Enzo Enabling Discovery in Life Science Lyso ID Green Detection Kit for microscopy Instruction Manual Cat No ENZ 51034 K500 500 assays For research use only Rev 1 0 May 2010 Notice to Purchaser The Lyso 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 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 purchase price All claims must be made
4. Reagent Quantity Lyso ID Green Detection Reagent Hoechst 33342 Nuclear Stain Chloroquine 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 diagnostic purposes The Lyso ID Green Detection Reagent contains DMSO which is read ily 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 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 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 Me
5. ces 1 Freundt EC Czapiga M and Lenardo MJ 2007 Photoconversion of Lysotracker Red to a green fluorescent molecule Cell Res 17 11 956 958 Nadrigny F Li D Kemnitz Ropert Koulakoff A Rudolph S Vitali M Giaume C Kirchhoff F and Oheim M 2007 Systematic colocali zation errors between acridine orange and EGFP in astrocyte vesicu lar organelles Biophys J 93 3 969 980 Jaiswal JK Fix M Takano T Nedergaard M and Simon SM 2007 Resolving vesicle fusion from lysis to monitor calcium triggered lysosomal exocytosis in astrocytes Proc Natl Acad Sci USA 104 35 14151 14156 Brunk UT Dalen H Roberg K and Hellquist HB 1997 Photo oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts Free Hadical Biology and Medicine 23 4 616 626 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 Vill Troubleshooting Guide Problem Potential Cause Suggestion Acidic organelles are not sufficiently stained Very low concentration of Lyso ID Green dye was used or dye was incubated with the cells for an insuffi cient 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 Lyso ID Green dye fails to stain a
6. cidic 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 bright Reduce the concentration of the nuclear counterstain or shorten the exposure time Untreated cells do not stain Cells do not appear healthy The lysosomal volume is small in untreated cells Some cells require serum to remain healthy Increase exposure time or add more dye Add serum to stain and wash solutions Serum does not affect staining Normal amounts of serum added range from 2 to 1096 Chloroquine treated cells appear dead or are no longer attached to the sur face The of chloroquine may be different with different cell lines Lower the dose of Chloro quine or reduce the time of exposure 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 F 610 941 9252 E info usaGenzoli
7. el B dyes All spectra were deter mined in 1X Assay Buffer 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 mitochondria within them They are typically spherical vesicles ranging in size from 0 2 to 2 um in diameter The lumen of lysosomes and other acidic organelles is characterized by low pH generated via proton pumping vacuolar ATPases Lyso ID Green 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 HeLa and U2OS cells treated with 300 uM chloroquine for 4 hours show a dramatic increase in lysosome like vesicle number and volume confirming Lyso ID Green dye is associated with this subcellular compartment Thus Lyso ID Green stain can be employed to highlight lysosome like organelles under certain condi tions 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 Vil Referen
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9. ge 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 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 FITC filter set for imaging the lysosomes Optionally image the nucleus using a DAPI filter set and the RFP tagged protein using a Texas Red filter set 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 Fluorescence Emission 500 Wavelength nm 600 700 Absorbance 200 250 300 350 400 450 500 550 600 Wavelength nm Fluorescence Emission Figure 1 Absorbance and fluorescence emission spectra for Lyso ID Green panel A and Hoechst 33342 pan
10. ng within a minute or two of dye addition The Lyso ID Green dye a new green emitting cell permeable small organic probe molecule that spontaneously localizes to live cell acidic organelles was developed to overcome the above problems It can be readily used in combination with other common UV and visible light excit able fluorescent dyes and various fluorescent proteins in multicolor imaging and detection applications The Lyso ID Green dye is suitable for both short term and long term tracking studies It emits in the FITC region of the visible light spectrum and is highly resistant to photobleaching concentra tion quenching and photoconversion The Lyso ID Green Detection Kit is specifically designed for use with RFP expressing cell lines as well as cells expressing blue cyan or orange fluorescent proteins BFPs CFPs OFPs A lysosome perturbation agent chloroquine is provided as a positive control for monitoring changes in lysosome number and volume A nuclear counterstain is also provided in the kit to highlight this organelle as well 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
11. ral 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 Dispense sufficient volume of Dual Detection Reagent see section V A3 page 3 to cover the monolayer cells 100 uL of labeling solution for cells grown on an 18 X 18 mm coverslip 3 Protect samples from light and incubate for 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 lysosomes Optionally image the nucleus using a DAPI filter set and the RFP tagged protein using a Texas Red filter set D STAINING LIVE CELLS GROWN IN SUSPENSION 1 Centrifu
12. thods 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 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 lysosomes 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 mmoles and should be centrifuged briefly to gather the material at the bottom of the tube Reconstitute the lyophilized material in 125 uL deionized water for a 60 mM stock solution It is recommended that treatment with the agent be performed using 10 300 uM 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
13. tions Lyso ID Green 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 Green dye are sufficient for staining mammalian cells This has been validated with the human cervical carcinoma cell line HeLa the human T lymphocyte cell line Jurkat and the human bone osteosarcoma epithelial cell line U2OS One important application of Lyso 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 associations and interac tions 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 metachromatic artifacts wherein fluorescent dyes emit both in the red and green regions of the spectrum compromise such analyses as observed with Acridine Orange dye These observations have led to spurious results in fluorescent protein co localization experiments 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 imagi
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