User Manual ENZ-51036-K025 - Cyto-ID Green Long
Contents
1. J ENZO Enabling Discovery in Life Science Cyto ID Green Long Term Cell Tracer Kit for flow cytometry and fluorescence microscopy Instruction Manual Cat No ENZ 51036 K025 For research use only Rev 1 0 1 June 2011 Notice to Purchaser The Cyto ID Green Long Term Cell Tracer 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 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 exploi tation 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 exten2. S 2 Apply 20 uL of the cell suspension to a glass microscope slide and overlay with a coverslip 3 Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC Green filter set for imaging the membrane signal ADHERENT CELLS 1 Seed stained cells at your desired density such as 1 x 107 cells mL Allow to adhere overnight Remove growth medium and add 1X HBSS step 3 page 3 as needed to maintain cell moisture and overlay with a coverslip 2 Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification is recommended Use a standard FITC Green filter set for imaging the membrane signal CELL ANALYSIS BY FLOW CYTOMETRY The protocol described in this manual assumes that the user is familiar with the basic principles and practices of flow cytometry and is able to run samples according to the operator s manual pertaining to the instrument being used 1 Collect stained cells at a density not to exceed 1X 10 cells mL NOTE Adherent cells will need to be trypsinized prior to analysis on the flow cytometer 2 Centrifuge at 400 x g for 5 minutes to pellet the cells Carefully suspend the cells in 500 uL of 1X HBSS 3 Read on bench top flow cytometer by gating out cellular debris and using a 488 nm blue laser for excitation and FL1 for signal regis tration VI APPENDICES A Filter Set Selection The selection of op
3. areful to remove as much liquid as possible during wash steps Organic based solvents or detergents were used that may extract the dye Use only aqueous based fixatives and solvents and limit the use of detergents Ka 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 usa 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 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 07621 5500 527 E info de enzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Frankrijklei 33 BE 2000 Antwerpen Belgium T 32 0 3 466 04 20 F 33 0 437 484 239 E info be enzolifesciences com www enzolifesciences com incorporating TERNATIONAL UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 0845601 1488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info uk enzolifesciences com www enzolifesc
4. e skin and may cause irritation to the eyes They should be treated as pos sible 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 To avoid photobleaching perform all manipulations in low light environments or protected from light by other means Methods are developed and optimized using kit components Use only supplied buffers for optimal results 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 2X Cyto ID Green Tracer Dye Solution IMPORTANT Prepare this reagent immediately before labeling cells In an appropriate size container mix by vortexing the following 2 uL Cyto ID Green Tracer Dye 1mL 1X Labeling Buffer from step A2 1X Labeling Buffer Dilute each milliliter mL of 4X Labeling Buffer with 3 mL deionized wate
5. gical tem peratures The kit is suitable for a variety of applications including long term cell viability cytotoxicity cell adhesion cell migration and cell cell fusion studies Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored upright at lt 20 C protected from light When stored properly these re agents are stable for one year upon receipt Avoid repeated freezing and thawing Reagents provided in the kit are sufficient for 25 reactions for flow cytome try or fluorescence microscopy Reagent Quantity Cyto ID Green Tracer Dye 4X Labeling Buffer 10X HBSS Additional Materials That May Be Required Flow cytometer equipped with 488nm blue laser Fluorescence microscope 15 ml and 50 ml conical tubes Adjustable speed centrifuge with a swinging bucket rotor CO incubator 37 C tissue culture plasticware tissue culture reagents 5 mL round bottom polystyrene tubes for holding cells Calibrated adjustable precision pipettors preferably with disposable plastic tips Glass microscope slides Glass cover slips Deionized water Serum e g Fetal Bovine Serum Growth medium eg Dulbecco s Modified Eagle Medium D MEM Safety Warnings and Precautions This product is for research use only and is not intended for diagnotic purposes Some components of this kit may contain hazardous substances Reagents can be harmful if ingested or absorbed through th
6. he Labeling Buffer 3 Prepare a 2X cell suspension by adding 1 mL of 1X Labeling Buffer to the cell pellet and suspending with gentle pipetting to insure complete dispersion Do not vortex and do not let cells stand in Labeling Buffer for periods longer than 15 20 min utes IMPORTANT The presence of physiologic salts like Ca and Mo causes the dye to form micelles and substantially reduces staining efficiency Therefore it is important that the cells be suspended in 1X Labeling Buffer pro vided at the time the dye is added not in medium or buffered salt solutions 4 Immediately prior to staining prepare a 2X Cyto ID Green Tracer Dye Solution in Labeling Buffer as described in step A1 on page 3 5 Rapidly add 1 mL of 2X cell suspension from step B3 above to 1 mL of freshly prepared 2X Cyto ID Green Tracer Dye solution and immediately mix well with a pipetor or equivalent to disperse IMPORTANT Staining is nearly instantaneous Brisk and consistent dispersion of the cells in the Cyto ID Green Tracer Dye solution is vital for consistent labeling 6 Incubate the cell dye suspension from step B4 for 2 5 minutes with periodic mixing Longer staining periods not to exceed 7 min utes will result in brighter cell staining NOTE Do not centrifuge the cells in Labeling Buffer before stopping the staining reaction 7 Stop the staining by adding an equal volume 2 mL of Stop Buffer and incubate for 1 minute to a
7. iences 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 33437 484 239 E info fr enzolifesciences com www enzolifesciences com Alexi S assay designs Stressgen
8. llow binding of excess dye Do not dilute with 1X Labeling Buffer 8 Centrifuge the cells at 400 x g for 5 minutes at 25 C and carefully remove the supernatant being sure not to remove cells To mini mize carryover of residual dye bound to the tube walls suspend cell pellet in 10 mL of complete serum containing medium trans fer to a fresh sterile conical polypropylene tube and centrifuge at 400 X g for 5 minutes at 25 C and wash the cell pellet 2 more times with 10 mL of complete medium each wash to ensure removal of unbound dye NOTE Staining efficiency is increased by transferring into a fresh tube at the first suspension step after staining Do not use Labeling Buffer for washing steps 4 9 After the final wash suspend the cell pellet in 10 mL of complete medium and transfer to a T25 flask or slides and incubate at 37 C and 5 CO overnight for no less than 12 hours to allow cells to recover 10 Perform cell counting and viability assessment After determining cellular recovery and viability the following steps may be per formed a Centrifuge the cell suspension and suspend to desired con centration of viable cells b Change the medium of the adherent cells CELL ANALYSIS By FLUORESCENCE CONFOCAL MicRoscoPY SUSPENSION CELLS 1 Collect the stained cells by centrifugation for 5 minutes at room temperature at 400 x g and carefully discard the supernatant Re suspend the cells with at least 20 uL of 1X HBS
9. mammalian cells and contains no detergents or organic solvents The appearance of labeled cells may vary depending upon the cell type from uniformly bright to punctuate This difference is thought to relate to the extent of membrane internalization occurring after cell labeling The Cyto ID Green Tracer dye fluores cence is independent of pH within normally encountered physiologic ranges and fluorescence intensity per cell is typically unaffected by the ultimate pattern of dye distribution The Cyto ID Green Tracer dye is not toxic to cells as determined using the benchmark MTT cell viability assay The dye is well retained by cells for up to 96 hours after loading and is passed to daughter cells upon mitosis Since the dye does not covalently modify proteins within the cells normal physiological responses are better preserved than with molecular probes based upon thiol reactive chloromethyl based or amine reactive succinimidyl ester based fluores cent dyes Dual labeling is also possible using a variety of available CELL estial dyes Labeled cells can be visualized by epifluorescence or confo cal fluorescence microscopy Additionally dye labeled and unlabeled cell populations can be analyzed by flow cytometry No transfer of fluores cence to adjacent cells was observed after a prolonged 96 hour incuba tion period This is in stark contrast to Calcein AM and BCECF AM which are only retained within viable cells for a few hours at physiolo
10. r 3 1X HBSS Allow the 10X HBSS to warm to room temperature Make sure that the reagent is free of any crystallization before dilution For every 10 mL of 1X HBSS needed dilute 1 mL of 10X HBSS with 9 mL deionized water Stop Buffer Prepare the Stop Buffer by adding 200 uL Fetal Bovine Serum FBS to 9 8 mL 1X HBSS from step A3 B STAINING LIVE SUSPENSION OR ADHERENT CELLS NOTE Cells are labeled by incorporating the dye into the cellular membrane Best results are a factor of cell concentration and dye concentration Loss of membrane integrity and poor cell recovery will result from over staining Also best results are obtained when adherent cells are dispersed into a cell suspension prior to staining Perform all subsequent steps at ambient temperature 20 25 C 1 Place a suspension containing 2 x 10 cells in a 15 mL conical bottom polypropylene tube Centrifuge cells at 400 x g and re move growth medium Wash twice with 4 mL 1X HBSS NOTE Serum proteins and lipids also bind the dye reducing the effec tive concentration available for cell labeling 2 Centrifuge the cells at 400 x g for 5 minutes into a loose pellet After centrifuging cells carefully discard the supernatant being careful not to remove any cells but leaving no more than 25 uL of supernatant IMPORTANT For reproducible results it is important to minimize the amount of residual medium or buffer present when cells are re suspended in t
11. rescence of mixed population of Jurkat cells over time Jurkat cells stained with Cyto ID Green Tracer dye were mixed with an unstained population of Jurkat cells and incubated over a 120 hour period Vil References 1 Horan P K and Slezak S E Stable Cell Membrane Labeling Na ture 340 167 168 1989 2 Horan P K et al Fluorescent Cell Labeling for in vivo and in vitro Cell Tracking Methods Cell Biol 33 469 490 1990 3 Poon R Y et al In Living Color Flow Cytometry and Cell Sorting Protocols Diamond R A and DeMaggio S eds Springer Verlag New York NY 2000 pp 302 352 4 Wallace P K and Muirhead K A Cell Tracking 2007 A proliferation of probes and applications Immunol Invest 36 527 562 2007 VIII Troubleshooting Guide Problem Potential Cause Suggestion Poor viability or recovery of cells Too much dye was incorpo rated into the cell mem branes Lower dye concentration and or increase cell num ber Cells remained in the label ing buffer too long Decrease the time that cells are in the labeling buffer The maximum time is 15 20 minutes Cross staining of cell popu lations Labeled cells lysed releas ing dye to adjacent cells Excess dye in solution or on the walls of the tube Lower dye concentration and or increase cell num ber Wash cells 3 5 times after labeling Transfer the sam ples to new tubes between washes Be c
12. t 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 Cyto 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 Le TREO GU CHI ON eege See init II Reagents Provided and Storage III Additional Materials That May Be Required IV Safety Warnings and Precautions V Methods and Procedures 1 2s0 rare A REAGENT PREPARATION nen B STAINING LIVE SUSPENSION OR ADHERENT CELLS C CELL ANALYSIS BY FLOURESCENCE CONFOCAL MICROSCOPY crt lata tir eed waste toh D CELL ANALYSIS BY FLOW CYTOMETRY ne VI Appendices rrrn eine A FILTER SET SELECTION i B EXPECTED RESULTS iii ANIL ReferencesS illr ara iniararne VIII Troubleshooting Guide Introduction The Cyto ID Green Long Term Cell Tracer Kit uses proprietary nonco valent cell labeling technology to stably incorporate a green fluorescent dye containing hydrophobic aliphatic chains into the cell membrane s lipid bilayer The dye may be loaded into cells by following the included proto col The labeling buffer is isotonic for
13. timal filter sets for a fluorescence microscopy applications 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 Use a standard FITC Green filter set for imaging the membrane signal 300 350 400 450 500 550 600 Figure 1 Fluorescence excitation 359 460 and emission 527 spectra for the Cyto ID Green Tracer dye All spectra were determined for cell bound dye B Expected Results Labeled cells can be visualized by epifluorescence microscopy using a standard FITC Green filter Figure 2 Dye labeled and unlabeled cell populations can be analyzed by flow cytometry No transfer of fluorescence to adjacent cells was observed after a prolonged 96 hour incubation period Figure 3 Figure 2 Composite bright field A and fluorescence microscopy images B demonstrating staining of Jurkat cells with Cyto ID Green Tracer dye Stan dard FITC Green filter set was used to image the membrane bound signal 6 OHr 24hr 48hr 120hr Time of Stained MFI Stained Unstained MFI Unstained Post Mixin Cells Cells Cells Cells 0 Hour 56 72 1466 11 40 47 2 97 24 Hours 52 92 451 65 44 39 4 08 48 Hours 50 63 101 84 46 59 3 91 120 Hours 51 59 12 3 46 23 3 24 Figure 3 Flow cytometry analysis of fluo
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