BTX ECM 2001 User Manual

Electrofusion systems

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ECM

2001 Electrofusion Systems

User's Manual

MA1 45-0080

MA1 45-0080INT

ECM

2001 Electro Cell Manipulator

(100 to 120 VAC)

ECM

2001 Electro Cell Manipulator

(200 to 230 VAC)

Publication 5501-002-REV-C

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Page 1 User’s Manual 2001 Electrofusion Systems ® MA1 45-0080 ® 2001 Electro Cell Manipulator (100 to 120 VAC) MA1 45-0080INT ® 2001 Electro Cell Manipulator (200 to 230 VAC) Publication 5501-002-REV-C...
Page 2 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).
Page 3: Table Of Contents
® 2001 Electrofusion/Electroporation Systems Table of Contents General Information: Serial Number ..............2 Calibration ..............2 Warranty ..............2-3 Service ..............3-4 Repair Facilities and Parts ..........4 General Safety Summary..........5-6 Electrical & Technical Specifications ......7-8 General Specifications............9 Introduction: Features.
Page 4: General Information
® 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...
Page 5: Service
This warranty gives you specific rights, and you may also have other rights, which vary from state to state. 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.
Page 6: Repair Facilities And Parts
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.
Page 7: General Safety Summary
® 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.
Page 8 ® 2001 Electrofusion/Electroporation Systems General Safety Summary (Continued) DO NOT OPERATE IN LOW IMPEDANCE Sample: Load or Sample If the electroporation samples have an impedance of less than 20 the samples may arc and result in sample loss and potential damage to unit.
Page 9: Electrical & Technical Specifications
® 2001 Electrofusion/Electroporation Systems Electrical & Technical Specifications Standard Capabilities: Operating Temperature 10˚ to 40˚C Storage Temperature 10˚ to 50˚C Humidity 90% relative humidity Altitude 2,000 m (operating) Pollution Degree 2 (Not to be operated in conductive pollutants atmosphere) 100 to 120 VAC, 50/60 Hz Power Requirements 200 to 230 VAC, 50/60 Hz Input Power...
Page 10 Less than a 10% V drop at the end of the pulse with the following constraints: PL ≤ 600 µs, load 3 40 BTX instruments are designed for in vitro and in vivo animal and plant applications only and are not for human Clinic. Page 8...
Page 11: General Specifications
® 2001 Electrofusion/Electroporation Systems General Specifications Certifications and Compliances Overvoltage Category: CAT III Products in this Category: Distribution-level mains, fixed installation CAT II: Local-level mains, applications and portable equipment CAT I: Signal levels in special equipment or parts of equipment, telecommunications and electronics Meets requirements of Directive 89/336/EEC for Electromagnetic Compatibility (EC) and Low-Voltage Directive 73/23/EEC for Product Safety.
Page 12: Introduction
® 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.
Page 13: Operation: Getting Started
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.btx@harvardapparatus.com) or your distributor.
Page 14 ® 2001 Electrofusion/Electroporation Systems Operation: Getting Started (continued) Location Requirements: • A sturdy, level, clean, non-flammable, dry surface • Adequate power source • Ambient room temperature of 15˚ to 40˚C • Relative humidity of 20 to 80%, non-condensing • A non-explosive atmosphere •...
Page 15: Quick Start
® 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.
Page 16 ® 2001 Electrofusion/Electroporation Systems Quick Start (Continued) # / Name Function 5. PULSE LENGTH 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”...
Page 17 ® 2001 Electrofusion/Electroporation Systems Quick Start (Continued) # / Name Function 12. MANUAL START 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.
Page 18: External Connections
® 2001 Electrofusion/Electroporation Systems Quick Start (Continued) External Connections: Output Terminals: Type SHV Coaxial Voltage Rating 5000V DC Max Remote Control: Type Mini-Din, 6-pos, Female Pinout Signal +12V Auto Start Manual Start Output Terminals AC Input and Power Switch Page 16...
Page 19: Replacing Power Entry Module Fuse
® 2001 Electrofusion/Electroporation Systems Quick Start (Continued) Replacing Power Entry Module Fuse Turn power switch to the “OFF” position Remove the Power Cable from the wall outlet ® Remove the Power Cable from the ECM 2001 Remove Fuse Holder Drawer (Refer to illustration) Remove both fuses and perform a visual inspection or a continuity test on each fuse Replace defective fuses with 8 A, 250 V, Time Lag...
Page 20: Operating Basics
9. Set Number of Pulses (7). 10. Set Post-Fusion AC Seconds (3) to the desired time period. 11. Load BTX Chamber with cell suspension/reagents. 12. Check all settings. 13. Press Automatic Start (9), a beep is emitted and the ®...
Page 21 ® 2001 Electrofusion/Electroporation Systems Operating 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.
Page 22 ® 2001 Electrofusion/Electroporation Systems Operating Basics (Continued) Operation With Microslides & 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.
Page 23: Advanced Operation: Programming
® 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. CAUTION Manual Switch Prior to turning on the ECM ® 2001, verify that the GREEN manual push button switch is in the "out"...
Page 24 ® 2001 Electrofusion/Electroporation Systems Advanced Operation: Programming (continued) Equipment Setup Setup of the ECM 2001 Independent of Monitoring ® Please refer to the following diagram for a graphical representation of the set up procedure for using the ECM ® 2001: 1.
Page 25: Experimental Methods
Ohnishi et al (BTX ECM ID# 218) and Ruzin (BTX ECM ID#,183) describe successful protocols for mammalian hybridoma production...
Page 26 ® 2001 Electrofusion/Electroporation Systems Experimental Methods (continued) 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.
Page 27 ® 2001 Electrofusion/Electroporation Systems Experimental Methods (continued) 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.
Page 28 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 Ce e l l l l P P r r e e p p a a r r a a t t i i o o n n 1.
Page 29 ® 2001 Electrofusion/Electroporation Systems Experimental Methods (continued) 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 10% NCS. 3. Expose oocyte pairs to KSOM containing cycloheximide (7.5 mg/ml), cytochalasin B (7.5 mg/ml), and 10% NCS for 1 hour to induce ooplast activation.
Page 30 ® 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.
Page 31: Appendix A: Troubleshooting
Make sure that the microscope is focused on Alignment cells between wires. Adjust accordingly. Increase the cell concentration if the cells move 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...
Page 32 ® 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. Fusion Did you set up the instrument right? START Check cable connection to microslide and verify Fusion...
Page 33 Observe if cells start to round off. Did you set up the instrument right? Change pre fusion treatment: were cells exposed Fusion to hypertonic solution. Call BTX - HARVARD APPARATUS at 1-800-272-2775 or 508-893-8999 (E-mail: techsupport.btx@harvardapparatus.com) or your distributor. Page 31...
Page 34 Verify cell viability using trypan blue. Reduce pronase/trypsin concentration and/or Division treatment time of cells. Post fusion care is critical for cell division. Division Verify procedure. Call BTX - HARVARD APPARATUS at 1-800-272-2775 or 508-893-8999 (E-mail: techsupport.btx@harvardapparatus.com) or your distributor. Page 32...
Page 35 If no, repeat experiment with myeloma cells in log growth phase. Is post fusion care proper? Ab Secretion Verify post fusion protocol. Ab Secretion Genetic makeup of hybrid could be incomplete. Call BTX - HARVARD APPARATUS at 1-800-272-2775 or 508-893-8999 (E-mail: techsupport.btx@harvardapparatus.com) or your distributor. Page 33...
Page 36 • 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”...
Page 37: Appendix B: Glossary Of Technical Terms
® 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.
Page 38 ® 2001 Electrofusion/Electroporation Systems Appendix B: Glossary of Technical Terms (continued) Dimer Formation The bringing together of two cells, through the process of dielec- trophoresis, so that they may be fused, resulting in a hybrid. Refer Dimer Formation also to Pearl Chain. 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...
Page 39 ® 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.
Page 40 ® 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.
Page 41: Appendix C: Accessories And Replacement Parts
® 2001 Electrofusion/Electroporation Systems Appendix C: Accessories and Replacement Parts Accessories Catalog Number Model Description MA1 45-0082 Cable Set, HV, 1 meter MA1 45-0083 Cable Set, HV, 3 meter MA1 45-0085 Cable, Remote Control, 2 meter MA1 45-0086 ECM2001R Remote Control Box MA1 45-0087 Adapter Set, Banana-to- Minigrabber...
Page 42 ® 2001 Electrofusion/Electroporation Systems Appendix C: Accessories and Replacement Parts (continued) Catalog Number Model Description MA1 45-0122 Genepaddles, 3 x 5 mm MA1 45-0123 Genepaddles, 5 x 7 mm MA1 45-0124 Cuvette, 1 mm Gap, pkg. 50 MA1 45-0125 Cuvette, 2 mm Gap, pkg. 50 MA1 45-0126 Cuvette, 4 mm Gap, pkg.
Page 43: Appendix D: General Cleaning And Maintenance
Reinstall filter mesh and outer cover. The ECM ® 2001 requires no maintenance. If the ECM ® 2001 does not work properly, contact BTX – Harvard Apparatus for appropriate instructions. Page 41...
Page 44: Ecm ® 2001 Generator Troubleshooting Guidelines
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.
Page 45 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. Page 43...

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Ma1 45-0080 Ma1 45-0080int