User`s Manual
Contents
1. Alignment Frequency 1 MEZ fixed Voltage 0 to 75 VRMS Duration 0 to 99 sec Post Fusion AC Frequency 1 MEZ fixed Voltage VRMS 1 10 of the alisnment AC amplitude attenuates with time Duration 0 to 9 sec Electroporation High Voltage Mode HV Mode Voltage 10 to 3000 V peak Pulse Length 1 to 99 psec Low Voltage Mode LV Mode Voltage 10 to 500 V peak Pulse Length 1 to 99 msec 0 01 to 0 99 msec Number of Repeats 0 to 9 Page 7 www btxonline com ECM 2001 Electrofusion Electroporation Systems Electrical amp Technical Specifications continued Note IV Mode Less than a 20 V drop at the end of the pulse with the following constraints PL x 25 ms load 3 20 O PL lt 1 sec load 3 1k Q PL lt 10 sec load 3 10k HV Mode Less than a 10 V drop at the end of the pulse with the following constraints PL lt 600 ps load 3 40 Q BTX instruments are designed for in vitro and in vivo animal and plant applications only and are not for human Clinic Page 8 www btxonline com ECM 2001 Electrofusion Electroporation Systems General Specifications CAUTION Certifications and Compliances FOR RESEARCH USE ONLY Overvoltage Category CAT III Products in this Category Distribution level mains fixed installation USE ON PATIENTS CAT II Local level mains applications and portable equipment CAT I Signal levels in special equipment or parts of equipment telecommunications and electronics Meets req2. Chambers Electroporation and Electrofusion Chambers are the devices used to contain the cells molecules to be fused transfected The chambers include the electrodes through which the 2001 pulse generator delivers the electric field to the cells Fusion Chambers include the Microslides Model 450 450 1 453 453 10 Meander Fusion Chamber Model 454 and the Flat Electrode Model 484 Cloning In terms of applications for electro cell fusion cloning refers to the ability to generate identical viable animals through processes such as nuclear transplantation Compression See Alignment Dielectrophoresis and Pearl Chains The result of an AC alignment Dielectric Breakdown The reversible breakdown of bi lipid layer membranes as a result of the application of a DC electroporation pulse A sufficiently high field strength may increase the membrane potential past a critical point leading to the breakdown of the membrane Dielectric Constant For a given dielectric nonmetallic material the ratio of electrical capacitance of a dielectric filled capacitor to a vacuum capacitor of identical dimensions Page 35 t E Alternating Current AC Disposable Electroporation Cuvettes Plus www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix B Glossary of Technical Terms continued Dimer Formation The bringing together of two cells through the process of dielec trophores
3. Service All service under the warranty will be made at the BTX HARVARD APPARATUS Holliston Massachusetts facilities or an authorized service site Owner will ship instrument prepaid to Holliston Massachusetts USA or the service site BTX HARVARD APPARATUS will return the instrument after servicing freight prepaid to owner s address Obtaining Service Service During Warranty 1 Write or call the BTX HARVARD APPARATUS Customer Support Group and describe the nature of the problem 2 Carry out minor adjustments or tests as suggested by BTX HARVARD APPARATUS 3 If proper performance is not obtained BTX HARVARD APPARATUS will notify you to ship the instrument prepaid to its Service Department The instrument will be repaired and returned at no charge for all customers in the continental United States Customers outside of the continental United States who have purchased our equipment from distributors should contact the distributor If you have purchased your equipment from us you should contact us directly We will repair at no charge but will not pay for shipment documentation etc These charges will be billed at cost Note Under no condition should the instrument or accessories be returned without prior approval from BTX HARVARD APPARATUS An RMA Returned Materials Authorization number must be obtained Page 3 www btxonline com ECM 2001 Electrofusion Electroporation Systems General Information
4. NOT FOR CLINICAL USE ON PATIENTS www btxonline com ECM 2001 Electrofusion Electroporation Systems General Safety Summary continved DO NOT OPERATE IN LOW IMPEDANCE Sample Load or Sample If the electroporation samples have an impedance of less than 20 O the samples may arc and result in sample loss and potential damage to unit Safety Terms and Symbols Terms that appear in this manual WARNING Warning statements identify conditions or practices that could result in injury or loss of life CAUTION Caution statements identify conditions or practices that could result in damage to these products or other property Symbols that may appear on the products AA Danger Attention Protective Functional High Refer to Earth Ground Voltage Manual Terminal Page 6 www btxonline com ECM 2001 Electrofusion Electroporation Systems Electrical amp Technical Specifications Standard Capabilities Operating Temperature 10 to 40 Storage Temperature 10 to 50 Humidity 9096 relative humidity Altitude 2 000 m operating Pollution Degree 2 Not to be operated in conductive pollutants atmosphere Power Requirements 100 to 120 VAC 50 60 Hz 200 to 230 VAC 50 60 Hz Input Power 85 W maximum idle 1 6 kW maximum peak Fusing T 8 A 250 V 5x 20mm Mechanical Characteristics Weight 22 kg 47 lbs Dimensions L x W XH 43 2 x 39 4 x 28 em 17 x 15 5 x 11 in Electrical Characteristics
5. idi m Call BTX HARVARD APPARATUS at 1 800 272 2775 or 508 893 8999 E mail techsupport btx harvardapparatus com or your distributor Page 31 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix A Troubleshooting continued Cells Do Not Divide Reduce the instrument settings for alignment amplitude and duration also the electroporation DC pulse amplitude and pulse length Verify cell viability using trypan blue START yes Division NO Reduce pronase trypsin concentration and or treatment time of cells yes Division NO Post fusion care is critical for cell division Verify procedure yes Division Call BTX HARVARD APPARATUS at 1 800 272 2775 or 508 893 8999 E mail techsupport btx harvardapparatus com or your distributor Page 32 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix A Troubleshooting continued Cells Do Not Secrete Antibody Ab Reduce instrument settings for alignment ampli tude and also the fusion amplitude Verify cell viability using trypan blue START YES Ab Secretion NO Reduce pronase trypsin concentration and or treatment time of cells ves Ab Secretion NO Immunization protocol might have been ineffective i e was animal hyperimmunized Change immunization YES Ab Secretion NO Were myeloma cells in log growth phase no repe
6. 035 V 25 sec DC FUSION 260 V 40 psec Yeild Total Fusions 8 6 Heterokaryons 2 6 Nuclear Transfer Cloning The following protocol is intended only as a guide to researchers involved in nuclear transfer cloning applications This protocol was adapted from Meng et al 1977 BTX ID 3588 ElectroSquarePorator T820 ELECTROFUSION PROTOCOL CELLS rhesus monkey oocyte Cell Preparation 1 Prepare enucleated recipient oocyte and donor blastomere as indicated in the paper 2 Prepare monolayer culture with buffalo liver cells BRL 1442 CMRL 1066 and 1096 neonatal calf serum Maintain monolayer culture at 6096 confluency during coculture and change medium every 24 hours Electroporation Settings Choose Mode HV 99msec 3 kV Set Voltage 130 to 140 V Set Pulse Length 50 msec Set Number of Pulses 2 pulses Chamber BTX microslide P N 450 0 5 mm gap Desired Field Strength 2 6 to 2 8 kV cm Page 26 www btxonline com ECM 2001 Electrofusion Electroporation Systems Experimental Methods continved Electroporation Procedure 1 Aspirate an individual blastomere into a microinjection pipette and place under the zona pelucida of an enucleated oocyte or zygote 2 Culture in KSOM with 1096 NCS 3 Expose oocyte pairs to KSOM containing cycloheximide 7 5 mg ml cytochalasin B 7 5 mg ml and 1096 NCS for 1 hour to induce ooplast activation Then place them individually in an electric field 4 Induce membrane fus
7. BTX HARVARD APPARATUS shall not be liable for any claims of any kind whatsoever as to the equipment delivered or for non delivery of equipment and whether or not based on negligence Warranty is void if the 2001 is changed any way from its original factory design or if repairs are attempted without written authorization by BTX HARVARD APPARATUS Warranty is void if parts connections or cell fusion chambers not manufactured by BTX HARVARD APPARATUS are used with the ECM 2001 Page 2 www btxonline com ECM 2001 Electrofusion Electroporation Systems General Information continued If a defect arises within the warranty period promptly contact BTX Harvard Apparatus 84 October Hill Road Building 7 Holliston Massachusetts USA 01746 1388 using our toll free number 1 800 272 2775 US Only or 508 893 8999 E mail techsupport btx harvardapparatus com Goods will not be accepted for return unless an RMA Returned Materials Authorization number has been issued by our customer service department The customer is responsible for shipping charges Please allow a reason able period of time for completion of repairs replacement and return If the unit is replaced the replacement unit is covered only for the remainder of the original warranty period dating from the purchase of the original device This warranty gives you specific rights and you may also have other rights which vary from state to state
8. Length 5 to 40 Set the DC Set Voltage 6 to 50 volts Set the Number of Pulses 7 to 2 Press the Automatic Start 9 button and release Do the process LEDs 14 illuminate Please contact BTX HARVARD APPARATUS Technical Services at 800 272 2775 or 508 893 8999 E mail techsupport btx harvardapparatus com if you answered no to any of the questions above Page 34 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix B Glossary of Technical Terms Alignment A consequence of cells being exposed to an inhomogeneous or diver gent electric field resulting in their movement toward the electrodes and subsequent alignment or pearl chain formation Alternating Current AC During electro cell fusion an alternating electrical current is utilized to induce an inhomogeneous or divergent electric field allowing for dielectrophoresis and pearl chain formation The resulting physical contact between cells facilitates the fusion process that may result fol lowing the application of a DC electroporation pulse Capacitor A device that stores energy in the form of an electric field A capacitor consists of two metal plates insulated from each other by a dielectric insulating usually a plastic material such as Mylar material In an ideal capacitor no conduction current flows between the plates after the capacitor is completely charged Capacitors can be fixed variable or adjustable
9. Programming continued Equipment Setup Setup of the 2001 Independent of Monitoring Please refer to the following diagram for a graphical representation of the set up procedure for using the ECM 2001 Ka HV Cable SCH Output Electrode ECM 2001 Accessory GENERATOR Remote Control Remote Control Box Accessory Optional 1 Connect the ECM 2001 into the appropriate line voltage outlet with the power cable 2 Connect electrode accessories to the HV output jacks on the rear of the unit using recommended HV cables and adapters See Appendix A 3 Proceed to Operation Page 22 www btxonline com ECM 2001 Electrofusion Electroporation Systems Experimental Methods Experimental Methods for Various Fusion Applications The following suggested procedure is a recommendation only and each investigator is encouraged to experiment with alternate cell den sities fusion media and pre and post fusion procedures and consult relevant publications Ohnishi et al BTX ECM ID 218 and Ruzin ECM 10 183 describe successful protocols for mammalian hybridoma production List of Materials Cells 1 Myeloma in log phase growth 2 Spleen peripheral blood cells etc Fusion Solutions Sterile 0 3 M mannitol glucose or related sugars 0 1 mM Ca and 0 1 mM Mg pH 7 0 to 7 3 adjust pH with 0
10. continued Out Of Warranty Service Proceed exactly as for Warranty Service above If our Service Department can assist you by phone or correspondence we will be glad to at no charge Repair service will be billed on the basis of labor and materials A complete statement of time spent and materials used will be supplied Shipment to BTX HARVARD APPARATUS should be prepaid Your bill will include return shipment freight charges Disassembly by the user is prohibited Service should only be carried out by experienced BTX HARVARD APPARATUS technicians Repair Facilities and Parts BTX Harvard Apparatus stocks replacement and repair parts When ordering please describe parts as completely as possible preferably using our part numbers If practical enclose a sample or drawing We offer complete reconditioning service Page 4 www btxonline com ECM 2001 Electrofusion Electroporation Systems General Safety Summary Please read the following safety precautions to ensure proper use of your ECM 2001 To avoid potential hazards and product damage use these products only as instructed in this manual If the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired To Prevent Hazard or Injury OBSERVE ALL TERMINAL RATINGS Review the operating manual to learn the ratings on all connections GROUND THE ECM 2001 This product is grounded through the g
11. the electric field Electrofusion EF or Electro Cell Fusion ECF Electric field induced cell fusion A novel physical means to bond two cells together by the application of an electric field pulse of high intensity Electrolytic A fluid containing charged molecules is called an electrolyte Electrolytic properties are associated with such a fluid such as the ability to conduct current Page 36 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix B Glossary of Technical Terms continued Electroporation The application of high electric field pulses of short duration to create temporary pores holes in the membranes of cells Embryo Manipulation The cloning of animals can be accomplished through embryo manipu lation techniques such as nuclear transfer and electrofusion Frequency The number of times an oscillation goes through a complete cycle in one second Unit is Hertz 1 sec Homogenous Electric Field The direction and field strength are constant Hybrid A viable daughter cell resulting from the fusion of two parent cells Hybridoma The fusion of an antibody producing cell with an immortalized cell resulting in an immortalized hybrid cell capable of generating mono clonal antibodies Hydrostatic Pressure The pressure in liquids at rest Inhomogeneous Electric Field Direction and strength of the electric field vary Microslide An electrofusion chamber allowing fo
12. the installation The location of the ECM 2001 should be a dry level surface free from extremes in ambient temperature dust or chemical exposures Provide a minimum of a 6 space between the back and any obstruction to airflow while a 2 space on both sides of the unit is required for adequate airflow Unpack cables chambers and accessories ordered and refer to OPERATION for further instructions Page 11 www btxonline com ECM 2001 Electrofusion Electroporation Systems Operation Getting Started continved Location Requirements A sturdy level clean non flammable dry surface Adequate power source Ambient room temperature of 15 to 40 C e Relative humidity of 20 to 80 non condensing Anon explosive atmosphere Adequate clearance for ventilation Lifting amp Carrying Handles are provided at each side of the front panel for lifting and carrying the unit Page 12 www btxonline com ECM 2001 Electrofusion Electroporation Systems Quick Start Front Panel Controls See Instrument above Name Function 1 SET AC VOLTAGE This knob adjusts the AC alignment voltage from 0 to 75 volts RMS The voltage is shown on the display to the left of the knob 2 AC DURATION The thumbpot controls the length of time that the SECONDS alignment is activated during the Automatic Mode The time is set using the and push buttons to select any time up to 99 seconds 3 POST FUSION AC The thumbp
13. 1 N NaOH Fusion Facilitators elective Trypsin at a concentration of 5 mg ml sterile without salt or Pronase at a concentration of 1 mg ml sterile without salt or Calmodulin 100 g ml and Glyceril mono oleate 50 pg ml Equipment amp Other Materials 1 RPMI 1640 or DMEM with 10 fetal bovine serum and 2 mM L glutamine 2 Trypan blue to determine viability of cells before and after fusion Sterile 96 microwell plate Sterile pipette Microscope with 10x and 40x objective Optional ice baths for reagents Laminar flow hood Table top centrifuge mp ap pt w Sterile conical tubes 10 Availability of Autoclave Suggested Pretreatment of Mouse Cells for ECF Spleen cells should be removed from the mouse and teased apart in cold Hank s Balance Salt Solution HBSS or RPMI 1640 Transfer spleen cells to a sterile test tube and allow the large particles to settle Pipette suspended spleen cells into a new sterile test tube Remove erythrocytes Preparations and cell concentrations for human cells and other fusion partners are the same Page 23 www btxonline com ECM 2001 Electrofusion Electroporation Systems Experimental Methods continved Fusion Protocol Establishment of optimal fusion parameters may take some time and must be done for each new myeloma lymphocyte pair Usually fusion is done at room temperature but as an alternative method one might elect to keep cell suspension on ice during processing Add
14. 10 V Push Manual Start button 12 and observe cells for 30 seconds If no movement occurs increase AC Voltage by increments of 10 V until movement is observed Page 27 www btxonline com ECM 2001 Electrofusion Electroporation Systems Experimental Methods continued Change cell suspension between experiments if heating or drying is observed Turn off the alignment signal if cell suspension is changed by pushing Manual Start 12 button 99 second limit a second time Signal is on as long as the AC waveform process LED is on continuously not flashing Experiment with AC Voltage and time period until a good percentage of the cells gently form dimers doublets within 10 to 20 seconds Note settings Set AC time period to the value found good for alignment Set the Post Fusion AC 3 time period up to 9 seconds to compress the cells after electrofusion Optimize system parameters with respect to cell viability Use the basic settings that were derived earlier and make several runs with modified settings Cultivate cells for 4 days from each run in RPMI 1640 or DMEM 10 fetal bovine serum hypoxanthine and thymidine 2 mM L glutamine and test part of the cell culture each day for viability by trypan blue exclusion Choose settings which lead to highest viability after 4 days System parameters which should be investigated for cell viability are as follows Vary Alignment Voltage and duration in steps of 2096 Hi
15. M 2001 Generator Troubleshooting Guidelines Basic Information Date Customer Name Institution Serial Number Software Version will be displayed as v x xx when you first power on the instrument Diagnostic Test Please complete the following chart Simply set the voltage pulse length pulse interval and pulse numbers on the ECM 2001 as indi cated and record the actual feedback after pulsing Use 400 pl of 1X PBS in a 4 mm cuvette as a 20 O load Use 200 pl of 1X PBS in a 4 mm cuvette as a 40 O load Make sure all cables are well connected and cuvettes make good contact with the safety stand to ensure prop er current flow ECM 2001 Manual Troubleshooting Section Please refer to Appendix A page 29 34 in the ECM 2001 manual for troubleshooting guidelines Please contact BTX Technical Service if the ECM 2001 routinely Arcs or if you smell anything coming from the machine Set Pulse Expected Expected Actual No v V PL PL 450 550 0 9 1 5 ms 450 550 90 110 ps 450 550 7 13 ps 850 1150 90 110 ps 2700 3300 90 110 ps Page 42 www btxonline com ECM 2001 Electrofusion Electroporation Systems ECM 2001 Generator Troubleshooting Guidelines continued Additional Information Please help us diagnose the problem by providing as much informa tion as possible 1 What is your main concern Please describe in detail Either instrument related or sample relate
16. User s Manual ECM 2001 Electrofusion Systems MAT 45 0080 ECM 2001 Electro Cell Manipulator 100 to 120 VAC MA1 45 0080INT 2001 Electro Cell Manipulator 200 to 230 VAC HARVARD APPARATUS The Electroporation Experts Publication 5501 002 REV C WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers We are committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for our products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of our products fall into either Category 8 or 9 and are c
17. at experiment with myeloma cells in log growth phase yes Ab Secretion NO Is post fusion care proper Verify post fusion protocol yes Ab Secretion NO Genetic makeup of hybrid could be incomplete yes Ab Secretion Call BTX HARVARD APPARATUS at 1 800 272 2775 or 508 893 8999 E mail techsupport btx harvardapparatus com or your distributor Page 33 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix A Troubleshooting continued Generator Test Generator Test Make sure the power cord is connected to the ECM 2001 and it is plugged in Turn on the power Do the displays illuminate Set the AC Voltage Setting 1 and DC Voltage Setting 6 to Zero Do the monitors display Zero Set Select Mode Knob 4 to the HV mode 1 to 99 psec Does the HV Mode green light illuminate Set Select Mode Knob 4 to the LV Mode 0 01 to 0 99 msec Does the LV Mode green light illuminate Set Select Mode Knob 4 to the LV Mode 1 to 99 msec Does the LV Mode green light illuminate Push in the Manual Start button Does the manual start button light illuminate Push in the Manual Start button again Does the manual start button light extinguish Set the following parameters on the instrument Set the AC Voltage 1 setting to 30 V Set the AC Durations 2 to 3 seconds Set the Post Fusion 3 to 3 seconds Set the Select Mode Knob 4 to HV Mode 1 to 99 usec Set the Pulse
18. ating Basics continued Selection of Operating Mode Automatic Start of the fusion process is initiated by pressing the Automatic Start button 9 in this case the pre set AC Voltage and AC Duration is applied Alignment is followed automatically by electro poration settings of DC PULSE Voltage and Pulse Length Manual Start Best with Microslides viewed under a microscope Push MANUAL START button 12 to turn on the alignment AC voltage It will illuminate in this position By pushing it in a second time the AC alignment voltage is turned off and electroporation pulses are delivered to the test chamber Alignment Parameters AC Voltage Dial the voltage setting required using the AC Voltage 1 AC Pulse Length Dial the AC Pulse Length on thumbpot AC Duration switch 2 Electroporation Parameters Choose between HV or IV Mode 4 Pulse Length Dial the pulse Length on DC Pulse Length Switch 5 DC Voltage Select the electroporation pulse Voltage by Set Voltage 6 Number of DC Pulses Set Number of pulses 7 Post Fusion AC Parameters Set the Post Fusion AC 3 time for compression of cells after electrofusion The post fusion AC amplitude is 1 10 of the alignment AC amplitude The post fusion AC amplitude attenuates with time Operation With Microslides amp Microscope To connect Microslides to the 2001 pulse generator attach Banana to Micrograbber Adapters to each HV cable and then connect the mic
19. ation 1 mp zappt w 11 12 13 14 Connect Chamber to the input located in the back panel of the ECM 2001 Plug the ECM 2001 power cable into correct line voltage outlet Switch POWER ON in the Back Panel Set AC Voltage 1 to desired voltage Set AC Duration Seconds 2 to desired time period Select between the HV or IV mode 4 Set DC Pulse Length 5 Set DC Voltage 6 Set Number of Pulses 7 Set Post Fusion AC Seconds 3 to the desired time period Load BTX Chamber with cell suspension reagents Check all settings Press Automatic Start 9 a beep is emitted and the 2001 delivers all preset parameters to the Chamber For electroporation only set AC Voltage 1 AC Duration 2 and Post Fusion AC 3 to ZERO Follow all other steps Manual Start Mode Operation 1 2 Page 18 Follow Steps 1to 13 in Automatic Operation Press Manual Start Button 12 manual start button illuminates and the AC waveform is output In this mode the AC Duration is over ridden and the AC field is on continuously until a 99 sec time out occurs or the button is released While viewing cells in a microslide under an inverted microscope the AC Amplitude may be varied to optimize alignment When cells are aligned appropriately press Manual Start Button again to release the AC and initiate the set DC parameters www btxonline com ECM 2001 Electrofusion Electroporation Systems Oper
20. d Please indicate past history for repairs and service if applicable 2 Please briefly outline your protocol below Cell line s Set voltage Set pulse length Set pulse number Set pulse interval Mode DN or HV circle one Buffer composition and volume Cell Density cell ml Transfectant Name ie DNA RNA protein etc Transfectant Amount Other information 3 Are you following an existing protocol or publication If so please specify Note If using BTX protocols please indicate the protocol ID number on the bottom left corner Final Instructions We appreciate your patience Please DO NOT send the instrument back to BTX without prior authorization from the company Instruments will not be received without a proper RMA Return Material Authorization number assigned When all the information is completed please contact BTX Technical Service via one of the following methods Email techsupport btx harvardapparatus com e 508 429 5732 Phone 1 800 272 2775 Thank you and we will do our best to assist you BTX Technical Service Group Page 43 www btxonline com
21. e Set HV 1 meter MAT 45 0083 Cable Set HV 3 meter MAT 45 0085 Cable Remote Control 2 meter MAT 45 0086 ECM2001R Remote Control Box MAT 45 0087 Adapter Set Banana to Minigrabber MAT 45 0088 Adapter Set Banana Splice F F MAT 45 0089 Adapter Set Banana to Square Post Cables MAT 45 0090 Adapter Set Banana to Pin Tip F F MA1 45 0207 630B Safety Stand MAT 45 0058 Enhancer Enhancer 2000 Electroporation 3000 Monitoring System MAT 45 0100 366 Petri Dish Electrode 2 mm Gap MAT 45 0101 384 Caliper Electrode 1 x 1 cm Brass MAT 45 0102 384L Caliper Electrode 1 5 x 1 5 cm amp 2x2 cm Stainless Steel MAT 45 0103 450 Microslide 0 5 mm Gap MAT 45 0104 450 1 Microslide 1 0 mm Gap MAT 45 0105 453 Microslide 3 2 mm Gap MAT 45 0106 453 10 Microslide 10 mm Gap MAT 45 0107 454 Meander Fusion Chamber MAT 45 0108 484 Flat Electrode 1mm Gap MAT 45 0109 485 Flatpack Chamber 1 83 mm Gap MAT 45 0110 486 Flatpack Chamber 0 56 mm Gap MA1 45 0113 508 Genetrode Straight 5 mm MAT 45 0114 510 Genetrode Straight 10 mm MA1 45 0115 512 Genetrode Bent L Shaped 5 mm MAT 45 0116 514 Genetrode Bent L Shaped 3 mm MAT 45 0117 516 Genetrode Bent L Shaped 1 mm MAT 45 0118 520 Tweezertrode Electrode 7 mm diameter MAT 45 0119 522 Tweezertrode Electrode 10 mm diameter MAT 45 0122 542 Genepaddles 3 x 5 mm MAT 45 0123 543 Genepaddles 5 x 7 mm MAT 45 0124 610 Cuvette 1 mm Gap pkg 50 MAT 45 0125 620 Cuvette 2 m
22. gher voltage requires shorter duration Vary DC Voltage and Pulse Length in steps of 2096 Experiment with Post Fusion AC duration to hold cells together after fusion Troubleshooting For trouble shooting refer to Appendix A Page 28 Cells Don t Align Cells Don t Fuse Cells Don t Divide Cells Don t Secrete Antibody www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix A Troubleshooting Cells Do Not Align Make sure the Microslide is clean free of cell debris Try varying the voltage while observing START dips the cells on the Microslide under microscope yes Alignment NO Make sure the equipment is set up right All cables should be connected ves Alignment NO Check the pH of the cell suspension It should be between 7 to 7 3 Adjust with 0 1 N NaOH If too much of NaOH is added this produces a basic con dition and produces undesired electrical field yes Alignment NO Are cells alive and in logarithmic growth phase If not change cells yes Alignment NO Make sure that the microscope is focused on cells between wires Adjust accordingly yes Alignment NO Increase the cell concentration if the cells move vts Alignment but do not align Call BTX HARVARD APPARATUS at 1 800 272 2775 or 508 893 8999 E mail techsupport btx harvardapparatus com or your distributor Page 29 www btxonline co
23. ion by 2 continuous square wave DC electrical pulses in 0 3 mannitol 100 mM 100 mM MgSO and 0 5 mg ml polyvinylpyrrolidone with the aid of a pipette controlled by a micromanipulator or just manually by hand 5 Oocyte pairs receive 2nd and 3rd pulses at hourly intervals Between pulses hold the pairs in KSOM cytochalasin B and cycloheximide 6 Reconstituted embryos are subsequently cocultured on a BRL cell monolayer Results 3 biochemical pregnancies resulted with one lost at 30 days of gesta tion The other two culminated in the births of one male and one female monkey Genetic typing indicated that the putative sire sperm donor and the putative mother female from which the nucleus was harvested were the true biological parents of both nuclear transfer infants Reference Meng et al Rhesus Monkeys Produced by Nuclear Transfer Biology of Reproduction 57 454 459 1997 DB 3588 Step By Step Procedures To Optimize Yield of Fusion Products After preparing the cell lines for fusion as described above the following procedures will aid in the determination of optimum instrument set tings This is important if new cell lines are to be fused for which no previous experimental data is available Determine instrument setting with Microslide Set both AC Voltage 1 and DC Voltage 6 to zero Set AC 2 and DC 5 time periods to zero kill wire gap with cell suspension e Increase the AC voltage to
24. is so that they may be fused resulting in a hybrid Refer also to Pearl Chain Dimer Formation Direct Current DC During electro cell fusion a direct current is utilized to produce a high intensity short duration electric field causing a reversible physical breakdown of bi lipid membranes and resulting in the formation of Direct Current DC temporary pores When juxtaposed pores in the membranes of two or more cells reseal following the DC pulse cells may become fused See Electric Field Frequency The number of times an oscillation goes through a complete cycle in one second The unit is either cycle sec or Hertz Hz Homogenous Electric Field An electric field where the direction and strength of the field lines are constant Divergence The deviation of field lines e g electric field lines from parallel homogeneous conditions A highly divergent field is a very inhomoge neous field where the value and direction of the field change drastically in the area under consideration Electric Field Electric Field Strength The potential difference between two points electrodes in Volts divided by the distance between the electrodes called gap and expressed in cm Expressed as V cm or kV cm This is true only if the electric field is homogenous as it is in parallel plate electrodes Electric Field Force The force acting on any charge in an electric field The force is equal to the product of the charge and
25. itionally one might decide to place fusion chambers in an ice bath during the actual fusion process Immediately prior to fusion the cells must be concentrated and washed in a nonelectrolyte solution Fusion In Standard 0 3 M Mannitol The two cell lines are combined at a 1 to 4 myeloma lymphocyte ratio and the mixture is centrifuged at 1 000 rpm for 6 minutes to obtain a cell pellet The supernatant is withdrawn and sterile 0 3 M mannitol with a pH between 7 to 7 3 is added The cells are washed two times in 0 3 M mannitol and resuspended to a final concentration of 2 x 106 cells per ml for fusion Cells in 0 3 M mannitol should not be allowed to remain in solution for more than 2 hours Therefore during the course of the experiment it may be necessary to prepare several cell pellets Fusion In Glucose As an alternative to standard 0 3 M mannitol one may elect to use other non electrolytic buffers such as 0 3 M glucose at a pH of 7 to 7 3 for fusion In this case one simply substitutes the sugar or other substance for the mannitol with the exception that the cells may be kept in the glucose for up to 4 hours Fusion Facilitators elective One might add pronase or trypsin to remove some of the membrane surface proteins and to neutralize possible surface electric charges This is advisable if cells are difficult to fuse Post Fusion Protocol for Standard 0 3 M Mannitol Glucose amp Related Sugars 1 Following the fusion proces
26. lectrolytic solution adjusting the density to 1 5 to 6 x 105 protoplasts ml according to size 4 Proceed with electrofusion Solutions In general optimal viability will occur when non electrolytic solutions are most potential 1 Page 25 consistent with pre fusion solutions in terms of osmotic pH and composition Non electrolytic sugars such as mannitol glucose sorbitol etc are recommended Low salt concentrations of CaCl 140 M and Hepes 600 M are optional and over a limited time do not appear to be detrimental pH settings can be explored for each protoplast type Fusions have been most successful at pH values of 7 0 to 7 5 Osmotic potential should be equal to the protoplast isolation solution Recalcitrant protoplasts have been successfully fused by the addition of two drops per ml of trypsin Sigma Typ XIII PCK treated 5 mg ml Other facilitators such as pronase dispase DMSO PEG etc should also be investigated www btxonline com ECM 2001 Electrofusion Electroporation Systems Experimental Methods continved Instrument Settings Typical fusion yields should average approximately 2096 with the addi tion of protease and 796 without PEG treated cells fuse typically at a 1 yield 1 Addition of more than 1 or 2 pulses appears to damage protoplasts without increasing yield 2 Typical settings for the Microslide might be as follows Amplitude Time Pulse Width AC ALIGNMENT
27. m ECM 2001 Electrofusion Electroporation Systems Appendix A Troubleshooting continued Cells Do Not Fuse Observe the microslide under microscope Fusion is successful if cell pairs do not drift apart after the fusion pulse and when they round off slowly START Did you set up the instrument right yes Fusion NO Check cable connection to microslide and verify continuity e g with an ohmmeter ves Fusion NO Increase fusion pulse amplitude and pulse length Cells will lyse at a critical upper setting Stay below this setting yes Fusion NO Do cells rotate If yes change the alignment Fusion amplitude alignment time NO Check pH of cell solution adjust to 7 to 7 3 using 0 1 N NaOH Were cells in fusion solution for extended period of time If yes use fresh cells in solution yes Fusion NO Add one drop of 1 mg ml pronase or one drop of 5 mg ml trypsin to the cell suspension about to be fused 30 sec before fusion yes Fusion NO GO TO THE NEXT PAGE Page 30 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix A Troubleshooting continued Cells Do Not Fuse continued Apply post fusion confinement force Observe if cells start to round off Fusion Did you set up the instrument right Change pre fusion treatment were cells exposed Fusion to hypertonic solution
28. m Gap pkg 50 Page 39 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix C Accessories and Replacement Parts continued Catalog Number Model Description MAT 45 0122 542 Genepaddles 3 x 5 mm MAT 45 0123 543 Genepaddles 5 x 7 mm MAT 45 0124 610 Cuvette 1 mm Gap pkg 50 MAT 45 0125 620 Cuvette 2 mm Gap pkg 50 MAT 45 0126 640 Cuvette 4 mm Gap pkg 50 MAT 45 0130 PP35 2P Petri Pulser 2 mm Gap MAT 45 0203 515 Genetrode Genepaddle Holder Spare Parts Catalog Number Model Description 06 004009 01 Page 40 Fuse T 8 A 250 V 5x 20 mm www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix D General Cleaning and Maintenance To clean exterior surfaces use a soft lint free cloth to remove loose dust For more efficient cleaning use a soft cloth dampened with water or an aqueous solution of 7596 isopropyl alcohol Do not immerse the ECM 2001 Avoid using abrasive cleaners Avoid using chemicals containing benzene or similar solvents To clean fan and vent filters remove outer cover and then remove filter mesh Rinse filter mesh with water to remove dust and allow to dry Reinstall filter mesh and outer cover The ECM 2001 requires no maintenance If the 2001 does not work properly contact BTX Harvard Apparatus for appropriate instructions Page 41 www btxonline com ECM 2001 Electrofusion Electroporation Systems EC
29. ot adjusts the AC duration after the SECONDS fusion pulse in the Automatic Mode Simply set the pulse length up to 9 seconds for the compression of cells after the fusion 4 SELECT MODE This knob selects the electroporation mode between the HV mode 10 to 3000 V 1 to 99 usec IV mode 10 to 500 0 01 to 0 99 msec ORIV mode 50 to 500 1 to 99 msec Once the selection is made the appropriate light will come on Page 13 www btxonline com ECM 2001 Electrofusion Electroporation Systems Quick Start Continued 3t Name 5 PULSE LENGTH 6 SET VOLTAGE 7 NUMBER OF PULSES 8 WAIT 9 AUTOMATIC START 10 REMOTE CONTROL 11 NUMBER OF REPEATS Page 14 Function The electroporation electrofusion pulse length thumbpot controls the pulse length of the squarewave pulse The pulse is set using the lower and upper push buttons to the ready mode This knob adjusts the pulse voltage If the generator is in the HV mode the voltage display left monitor is in Volts and it can be set from 10 to 3000 V in the HV Mode and 10 to 500 in the LV Modes Turn clockwise to increase counter clockwise to decrease the voltage Set number of pulses from 1 to 9 The and push buttons are used to select the desired number of pulses The yellow light illuminates when each pulse is delivered to the chamber and a beep is emitted This light illuminates when the instrument has reached the end of an opera
30. r the observation of the align ment and fusion processes under a microscope Osmotic Pressure The applied pressure required to prevent the flow of solvents of differ ent concentration across a semipermeable membrane Pearl Chains See Alignment and Dielectrophoresis Chains of cells or vesicles brought into alignment during electro cell fusion prior to electroporation Pore A small mostly transient opening in a cell membrane wall caused by the application of a brief high electric field pulse Page 37 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix B Glossary of Technical Terms continued Potential Difference The difference in Volts between points in an area between electrodes Pressure Gradient The difference in pressure between two points in a medium Pulse Length The length of time an electric signal is applied Relaxation Time The time of a system to reach equilibrium Rounding Off The phenomena of cells forming a sphere after being fused together Turgor Pressure The pressure in capillaries Voltage The difference of electric potential between two electrodes expressed in volts V or kilovolts kV Wave Forms The shape of time varying electric signals Page 38 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix C Accessories and Replacement Parts Accessories Catalog Number Model Description MAT 45 0082 Cabl
31. rating at low impedance loads of 20 Page 10 www btxonline com ECM 2001 Electrofusion Electroporation Systems Operation Getting Started Unpacking The ECM 2001 Electro Cell Manipulator System is shipped in a carton specifically designed to provide maximum protection for the instrument during transportation under normal handling conditions Upon receipt open the carton and carefully remove the ECM 2001 The contents should be examined for any apparent damage resulting from shipment and if necessary BTX HARVARD APPARATUS should be informed in writing Be sure to save the carton and packing materials for any future transportation and shipping requirements Packing Data Check the packing slips to ensure that all items ordered and listed are included in the shipment If any parts are missing call BTX HAR VARD APPARATUS at 1 800 272 2775 US Only or 508 893 8999 E mail techsupport btxGharvardapparatus com or your distributor CAUTION Power Source As received the ECM 2001 is ready for use with either a 100 to 120 VAC or 200 to 230 VAC single phase 50 60 Hz power source according to the electrical requirements of the country to which it is shipped The power cord has a standard three prong plug For safety reasons it is mandatory that the instrument be grounded DO NOT MODIFY THE PLUG Installation Once you have determined that none of the ECM 2001 components are damaged or missing proceed with
32. rograbbers to the microslide contacts It is advisable to tape the cables to the microscope stand to avoid the weight of the cables moving the Microslide The Microslide wire electrode gap can be filled and emptied with a micropipette tip or a syringe with a hypodermic needle Page 19 www btxonline com ECM 2001 Electrofusion Electroporation Systems Operating Basics continued Operation With Microslides amp Microscope continued Adjust the AC voltage setting so that cells move slowly toward the Microslide Electrodes Increase fusion voltage and pulse length in small increments 10 to get increased fusion yield Cells will drift apart after alignment if the fusion pulse is absent or too weak Look for fusion events close to the wires The electric field is strongest close to the wires and fusion will occur here first If the field is too strong the cells will burst lyse immediately If the cells do not move and an increase in voltage leads to boiling of the media indications are that the cell solution acts too much like an electrolyte Use fresh mannitol to prepare 0 3 M mannitol 0 2 micron filtered with a pH of 7 0 to 7 3 Adjust pH with sterile 0 1 N NaOH Measure pH of mannitol just prior to fusion Other low molecular weight sugars such as sucrose and glucose have also been used successfully When operating Microslides under a microscope it is advantageous to use the generator in the Manual Mode This way DC fusion p
33. rol Type Mini Din 6 pos Female Pinout Pin Signal 1 2 RTN 3 12V 4 Auto Start 5 Manual Start 6 NIC Output Terminals AC Input and Power Switch Page 16 www btxonline com ECM 2001 Electrofusion Electroporation Systems Quick Start Continued Replacing Power Entry Module Fuse 1 Turn power switch to the OFF position Remove the Power Cable from the wall outlet Remove the Power Cable from the ECM 2001 2 3 4 Remove Fuse Holder Drawer Refer to illustration 5 Remove both fuses and perform a visual inspection or a continuity test on each fuse 6 Replace defective fuses with 8 A 250 V Time Lag Slo Blo 5 x 20 mm Fuses Refer to Spare Parts pg 32 for ordering information 7 Install Fuse Holder Drawer refer to illustration 8 Install the Power Cable into the ECM 2001 9 Plug Power Cable into wall outlet 10 Turn power switch to the ON position H B FUSE DRAWER 86 10 06 1 De Additional Fusing Not Operator Replaceable Fuse Rating T 0 1A 250V T 0 1A 250V T 1 6A 250V T 2 0A 250V T 3 15A 250V T 3 15A 250V 250V Page 17 Location XFMR T1 Secondary 2 XFMR T1 Secondary 4 XFMR T1 Secondary 1 XFMR T1 Secondary 3 XFMR T2 Primary 1 XFMR T2 Primary 2 XFMR 4 Secondary www btxonline com ECM 2001 Electrofusion Electroporation Systems Operating Basics Automatic Start Mode Oper
34. rounding conductor of the power cord To avoid electric shock the grounding conductor must be connected to earth ground Before making any connections to the input or output terminals of the product ensure that the product is properly grounded MAKE PROPER CONNECTIONS Make sure all connections are made properly and securely DO NOT OPERATE WITHOUT COVERS To avoid electric shock or fire hazard do not operate these products with the covers removed AVOID EXPOSED CIRCUITRY Do not touch exposed connections when power is present USE PROPER LINE CORD Use only the specified line cord for this product and make sure line cord is certified for country of use USE PROPER FUSE Use only specified fuses with product DO NOT OPERATE WITH SUSPECTED FAILURES If damage is suspected on or to the products do not operate Contact qualified service personnel to perform inspection DO NOT OPERATE IN EXPLOSIVE ATMOSPHERE To avoid injury or fire hazard do not operate these products in an explosive environment DO NOT OPERATE IN WET DAMP CONDITIONS To avoid electric shock do not operate these products in wet or damp conditions OBSERVE ALL WARNING LABELS ON PRODUCT Read all labels on the products to ensure proper usage DO NOT CONTINUALLY PULSE UNIT If you continue to pulse the unit without a 10 20 second delay between pulse series the unit may overheat and result in damage to components Page 5 CAUTION FOR RESEARCH USE ONLY
35. s remove cell suspension under sterile conditions from the fusion chamber and carefully pipette into a sterile test tube Add 10 ml of RPMI 1640 or DMEM with 10 fetal bovine serum and 2 mM L glutamine Allow to rest for 15 minutes 2 Centrifuge cells at 500 rpm for 5 minutes and remove supernatant Carefully resuspend cells in culture medium and pipette cells into a 96 microwell plate at a concentration of 2 x 105 cells per well 0 2 ml per well Check wells for sufficient amount of medium 3 Incubate cells at 37 with 596 CO overnight Page 24 www btxonline com ECM 2001 Electrofusion Electroporation Systems Experimental Methods continved 4 The following day remove one half of the medium and re feed cells with HAT medium On days 4 7 and 10 again remove one half of the medium and re feed with HAT Cells may be supplemented with feeder layers or other growth factors Check cultures daily for HAT medium selection Once colony formation is observed begin feeding with HAT medium at first phase of expansion Plant Protoplast Fusion The following protocol is intended only as a guide to researchers involved in the genetic engineering of plant protoplasts Basic Procedure 1 Obtain freshly isolated protoplasts as per usual methods Note Fusion will not be successful after cell wall regeneration has occurred 2 Gently pellet in non electrolytic solution 3 Remove supernatant and resuspend in non e
36. ting cycle and discharges all the capacitors for safety The generator cannot be operated when this light is on CAUTION Do not change settings or initiate another cycle until wait light has extinguished Press and release to initiate an alignment and electroporation cycle according to the preset voltage pulse length and number of pulses Optional Accessory Connect the remote control box cable so the generator can be operated from the remote control box The thumbpot can be used to set the number of repeat cycles It can be set up to 9 cycles www btxonline com ECM 2001 Electrofusion Electroporation Systems Quick Start Continued Name 12 MANUAL START 13 PROCESS LEDS Page 15 Function This two position button allows manual control of the ECM 2001 Push IN to Start the alignment The button illuminates and the alignment voltage is applied continuously until the button is pushed a second time to release or a timeout of 99 sec is reached When released the green light goes out and the electrofusion pulses are delivered to the chamber If the timeout is reached the AC waveform process LED changes from continuously on to flashing These LEDs illuminate to indicate each stage of the process www btxonline com ECM 2001 Electrofusion Electroporation Systems Quick Start Continued External Connections Output Terminals Type SHV Coaxial Voltage Rating 5000V DC Max Remote Cont
37. uirements of Directive 89 336 EEC for Electromagnetic Compatibility EC and Low Voltage Directive 73 23 EEC for Product Safety Compliance was demonstrated to the following specifications as listed in the Official Journal of the European Communities EN 50081 1 Emissions EN 55011 Class B Radiated and Conducted Emissions EN 55082 1 Immunity IEC 10004 2 Electrostatic Discharge Immunity IEC 10004 3 RF Electromagnetic Field Immunity IEC 10004 4 Electrical Fast Transient Burst Immunity Low Voltage Directive 73 23 EEC EN 61010 1 Safety requirements for electrical equipment for measurement control and laboratory use NOT FOR CLINICAL Page 9 www btxonline com ECM 2001 Electrofusion Electroporation Systems Introduction The ECM 2001 is a multifunctional electrofusion and electropo ration square wave generator Embryo and cell fusion are facilitat ed by the combination of the AC and DC wave pulses Fusion is achieved by the generation of an AC current wave form that gen erates a benign dielectrophoretic alignment of cells With only microsecond switchover time from AC to DC efficient fusion takes place After fusion the AC is reapplied maintaing the cell compression for the rounding off process resulting in a higher number of hybrids ECM 2001 features include AC waveform of 1 MHz optimal for fusion Electroporation capabilities e Awide range of voltages from 5 to 3000 V e Finer voltage discrimination Capable of ope
38. ulses can be initiated as soon as alignment is achieved thus subjecting the cells to minimal electrical stress Use a stopwatch to measure the required alignment time Set the same time or longer in the automatic mode when working with larger chambers If a demonstration for several people is intended a microscope with a TV camera attachment and monitor display serves well Page 20 www btxonline com ECM 2001 Electrofusion Electroporation Systems Advanced Operation Programming Important Notices Please refer to the following important notices prior to setting up and using the ECM 2001 A CAUTION Manual Switch Prior to turning on the ECM 2001 verify that the GREEN manual push button switch is in the out or Off position and that it is not pushed in If the instrument power is turned on and the GREEN manual switch light comes on turn it off immediately Only use the manual mode with appropriate buffers under the appropriate conditions for AC alignment optimization A WARNING Procedure WARNING HIGH VOLTAGE For practical and safety reasons do not touch any of the cables or electrode connections while the 2001 is in the Manual Start or Automatic Start Modes and the AC field or DC pulses is in use Check that the unit is not operating and is in standby mode when connecting and disconnecting cables Page 21 www btxonline com ECM 2001 Electrofusion Electroporation Systems Advanced Operation
39. urrently exempt from the RoHS Directive We will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as they apply Do Not Dispose Product with Municipal Waste Special Collection Disposal Required ECM 2001 Electrofusion Electroporation Systems Table of Contents General Information Serial Number cn eterne inen 2 EE 2 Vol EE 2 3 SEU VICS eases 3 4 Repair Facilities and 4 General Safety Summary es Electrical amp Technical Specifications General Introduction e TE 10 Operation Getting Started 11 12 Quick Start Front Panel Controls een External Connections En Replacing Power Entry Module Operating Basics eere Advanced Operation Programming Experimental Methods eee Appendix A Troubleshooting Appendix B Glossary of Technical Terms 36 39 Appendix C Accessories and Replacement Parts 40 41 Appendix D General Cleaning and Maintenance 42 ECM 2001 Generator Troubleshooting MINIM 43 44 Page 1 www btxonline com ECM 2001 Electrof
40. usion Electroporation Systems General Information Serial Number The serial number for the ECM 2001 is located on the rear of the instrument case All inquiries concerning these products should refer to the serial numbers on the units Calibration There is no calibration required for the ECM 2001 Warranty BTX Harvard Apparatus warranties the ECM 2001 for a period of two years from the date of purchase At its option BTX Harvard Apparatus will repair or replace the unit if it is found to be defective as to workmanship or materials This warranty does not extend to any instrumentation which has been a subjected to misuse neglect acci dent or abuse b repaired or altered by anyone other than BTX HARVARD APPARATUS without BTX HARVARD APPARATUS express and prior approval c used in violation of instructions fur nished by BTX HARVARD APPARATUS This warranty extends only to the original customer purchaser IN NO EVENT SHALL BTX HARVARD APPARATUS BE LIABLE FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclusion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied warranty so the above limi tation may not apply to you Without limiting the generality of the foregoing
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