User`s Manual
Contents
1. Warning Use proper eye protection during electroporation 6 Remove the tissue carefully clean as appropriate and prepare for the next experiment 2 Needle Array 1 Grasping Model 530 or Model 532 2 Needle Array Handle position the handle over a Model 531 or Model 533 2 Needle Array Assembly and push to secure the 2 Needle array to the handle 2 Push the banana plugs at the opposite end of the 2 Needle array handle into the voltage output of the BTX generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 Then plug the banana cable into the voltage output of the BTX Generator 3 Following instructions for the BTX generator set the appropriate parameters 4 Prepare tissue and sample for electroporation Apply sample to tissue just before electroporation 5 Remove the safety shield protecting the needles place into the tissue and deliver the electroporation pulse s Warning Use proper eye protection during electroporation 6 Discard the 2 needle array and prepare for the next experiment Microslides 1 Push the banana plugs at the opposite end of the micrograbber cable catalog number 45 0216 into the voltage output of the BTX generator Warni2. 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 requirements 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 ac
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 process 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 C with 5 CO overnight Page 24 www btxonline com ECM 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
4. NOT OPERATE WITH SUSPECTED FAILURES If damage is suspected on or to the products do not operate Contact qualified BTX 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 Page 5 CAUTION FOR RESEARCH USE ONLY NOT FOR CLINICAL USE ON PATIENTS www btxonline com ECM 2001 Electrofusion Electroporation Systems General Safety Summary continued 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 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 Standar
5. 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 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 Then place them individually in an electric field 4 Induce membrane fusion by 2 continuous square wave DC electrical pulses in 0 3 mannitol 100 mM CaCl 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 Nucl
6. PN286 0 66 as Loe J om g e ee Pe En S FUSE DRAWER ear il wa mm W Additional Fusing Not Operator Replaceable Fuse Rating Location T 0 1A 250V XFMR T1 Secondary 2 T 0 1 A 250V XFMR T1 Secondary 4 T 1 6A 250V XFMR T1 Secondary 1 T 2 0 A 250V XFMR T1 Secondary 3 T 22 T 3 1 5A 250 V XFMR T2 Primary 1 5A 250 V XFMR T2 Primary 2 T 4A 250V XFMR T4 Secondary Page 17 www btxonline com ECM 2001 Electrofusion Electroporation Systems Operating Basics Automatic Start Mode Operation 1 mp zappt w n 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 LV 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 ECM 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 butto
7. 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 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 d
8. 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 x L S i 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 trophoresis 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 aw 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 hom
9. 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 for 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 o
10. 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 4 Obtain freshly isolated protoplasts as per usual methods Note Fusion will not be successful after cell wall regeneration has occurred Gently pellet in non electrolytic solution Remove supernatant and resuspend in non electrolytic solution adjusting the density to 1 5 to 6 x 105 protoplasts ml according to size Proceed with electrofusion Solutions In general optimal viability will occur when non electrolytic solutions are most consistent with pre fusion solutions in terms of osmotic potential 1 Page 25 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 T
11. the BTX generator set the appropriate parameters Place the Genetrodes Genepaddles on the sample using a micromanipulator or manually position them Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation Remove the electrodes carefully clean as appropriate and prepare for the next experiment Tweezertrodes 1 Page 43 Attach the Model 524 Tweezertrode Cables to the electrode base of the Tweezertrode Push the banana plugs at the opposite end of the tweezertrode cable into the voltage output of the BTX generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 Then plug the banana cable into the voltage output of the BTX Generator Following instructions for the BTX generator set the appropriate parameters www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix F Connecting Electrodes continued 4 Prepare tissue and sample for electroporation Grasp the tissue between the Tweezertrode electrodes and measure the interelectrode distance Adjust generator settings if necessary Inject the sample into the tissue 5 Deliver the electroporation pulse s to the sample
12. 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 20 Higher voltage requires shorter duration Vary DC Voltage and Pulse Length in steps of 20 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 ees 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
13. 0 Appendix E ECM 2001 Footswitch Compatibility 40 Appendix F Connecting Electrodes sssses0e 41 46 ECM 2001 Generator Troubleshooting ITT TE 47 48 Page 1 www btxonline com ECM 2001 Electrofusion 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 Failure to use the Enhancer 3000 High Voltage probe to connect a BTX Generator to an external digital oscilloscope for monitoring will result in voiding your warranty connecting directly to the external monitoring equipment or modified monitoring setup will damage the Generat
14. 001 does not work properly contact BTX Harvard Apparatus for appropriate instructions Appendix E ECM 2001 Footswitch Compatibility There are two models of footswitches for the ECM 2001 In 2005 the ECM 2001 was redesigned for CE compliance and approval These changes resulted in a modification to the footswitch output To determine which footswitch is compatible with your generator please locate the serial number on the rear panel of the generator If the serial number of your ECM 2001 Generator is gt 4000 then you will use the CE Marked Footswitch product number 45 0085 If the serial number of your ECM 2001 Generator is lt 3999 then you will use the Non CE Marked Footswitch product number 45 0214 Page 40 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix F Connecting Electrodes Cuvettes Safety Stand 1 Remove the cuvette and the transfer pipette from their sterile packaging 2 Remove the cuvette cover and fill the cuvette with sample using the transfer pipette and replace cover 3 Place cuvette on ice for cooling purposes 4 Push the banana plugs of the Safety Stand into the voltage output of the BTX generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect t
15. REEN 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 ECM 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 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 Ka TH Power In RES HV Cable Output ee ECM 2001 ccessory 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 F
16. Remove the Petri Pulser carefully clean as appropriate and complete delivery of pulses to all wells if using a 6 well plate Caliper Electrodes 1 Attach the Caliper Electrodes directly to the voltage output ports of the BTX Generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 2 Prepare tissue for electroporation 3 Following instructions for the BTX generator set the appropriate parameters 4 Use the Caliper Electrodes to span the target tissue 5 Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation Do not exceed 500V 6 Remove the Caliper Electrodes carefully clean as appropriate and prepare for next experiment Page 46 www btxonline com ECM 2001 Electrofusion Electroporation Systems ECM 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 feedba
17. User s Manual ECM 2001 Electrofusion Systems ee ECM 2001 Electro Cell Manipulator 45 0080 ECM 2001 Electro Cell Manipulator 100 to 120 VAC 45 0080INT ECM 2001 Electro Cell Manipulator 200 to 230 VAC HARVARD APPARATUS The Electroporation Experts Publication 5501 002 REV E 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 instru ments Most of our products fall into ei
18. c 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 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 ECM 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
19. ck after pulsing Use 400 pl of 1X PBS in a 4 mm cuvette as a 20 Q load Use 200 pl of 1X PBS in a 4 mm cuvette as a 40 Q 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 Expected Expected vv 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 47 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 related 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 LV 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 prot
20. ctroporation 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 ECM 2001 pulse generator attach Banana to Micrograbber Adapters to each HV cable and then connect the micrograbbers 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 increas
21. d 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 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 5x20 mm Mechanical Characteristics Weight 22 k 47 lbs Dimensions L x W x H 43 2 x 39 4 x 28 cm 17 x 15 5 x 11 in Electrical Characteristics Alignment Frequency 1 MHz fixed Voltage 0 to 75 VRMS Duration 0 to 99 sec Post Fusion AC Frequency 1 MHz fixed Voltage VRMS 1 10 of the alignment 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 to9 Page 7 www btxonline com ECM 2001 Electrofusion Electroporation Systems Electrical amp Technical Specifications continued Note LV Mode Less than a 20 V drop at the end of the pulse with the following constraints PL lt 25 ms load 3 20 Q PL lt 1 sec load 3 1k Q PL lt 10 sec load 3 10k Q 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
22. e 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 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 Additionally 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
23. e 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 pulses 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 Note The ECM 2001 Electrofusion Electropation system is NOT compatible with the HT highthroughput plate handlers 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 G
24. e parameters 5 Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation 6 Extract the cell solution The chamber can be rested on ice for cooling purposes 7 Clean as appropriate and prepare for the next experiment Page 42 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix F Connecting Electrodes continued Genetrodes Genepaddles 1 Using the Model 515 Genetrodes holder loosen the two plastic screws and separate the top half of the positioning plate from the holder Place the pair of electrodes in the predetermined slots based on the necessary gap size The electrodes must extend from the holder in the opposite direction of the holder handle Secure the electrodes by reassembling the holder and tightening the two screws Attached the micrograbber cable catalog number 45 0216 to the electrode leads of the Genetrodes Genepaddles Push the banana plugs at the opposite end of the micrograbber cable into the voltage output of the BTX generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 4 Prepare tissue and sample for electroporation Following instructions for
25. ear 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 e Fill wire gap with cell suspension Increase the AC voltage to 10 V Page 27 www btxonline com ECM 2001 Electrofusion Electroporation Systems Experimental Methods continued 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 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
26. f 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 o 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 45 0082 Cable Set HV 1 meter 45 0083 Cable Set HV 3 meter 45 0085 Cable Remote Control 2 meter 45 0086 ECM2001R Remote Control Box 45 0087 Adapter Set Banana to Minigrabber 45 0088 Adapter Set Banana Splice F F 45 0089 Adapter Set Banana to Square Post Cables 45 0090 Adapter Set Banana to Pin Tip F F 45 0207 630B Safety Stand 45 0059 Enhancer Enhancer 2000 Electroporation 3000 Monitoring System 45 0100 366 Petri Dish Electrode 2 mm Gap 45 0101 384 Caliper Electrode 1 x 1 cm Brass 45 0102 384L Caliper Electrode 1 5 x 1 5 cm amp 2 x 2 cm Stainless Steel 45 0103 450 Microslide 0 5 mm Gap 45 0104 450 1 Microslide 1 0 mm Gap 45 0105 453 Microslide 3 2 mm Gap 45 0106 453 10 Microslide 10 mm Gap 45 0107 454 Meander Fusion Chamber 45 0108 484 Flat Electrode 1mm Gap 45 0109 485 Flatpack Chamber 1 83 mm Gap 45 0110 486 Flatpack Chamber 0 56
27. he BTX generator to the input ports of the Enhancer 3000 5 Open the Safety Stand cover 6 Use the thumb wheel to slide the electrodes open Place a BTX cuvette in between the electrodes with the aluminum of the cuvette coming in contact with the electrodes 8 Secure the cuvette in place by closing the gap with the thumb wheel The cuvette should fit snugly between the electrodes however it should be loose enough that it can be pulled out without adjusting the thumb wheel again 9 Following instructions for the BTX generator Set the appropriate parameters 10 Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation 11 Remove cuvette cover and extract the cell solution 12 Dispose of cuvette and prepare for the next experiment Flat Pack Chambers 1 Fill the flat pack chamber with sample using a pipette 2 Push the banana plugs of the Safety Stand into the voltage output of the BTX generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 3 Open the Safety Stand cover Page 41 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix F Connecting Electrodes contin
28. he input ports of the Enhancer 3000 2 Prepare sample for electroporation a 35mm Petri Dish 3 Place the electrode in the 35mm Petri Dish Allow it to gently rest on the surface of the dish 4 Following instructions for the BTX generator set the appropriate parameters Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation 5 Remove the Petri Dish Electrode carefully clean as appropriate and prepare for next experiment Petri Pulser 1 Plug the HV cables from the Petri Pulser into the voltage output of the BTX Generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 2 Prepare sample for electroporation in 6 well plate or in 35mm Petri Dish Page 45 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix F Connecting Electrodes continued 3 Place the electrode in one well of the 6 well plate or 35mm Petri Dish Allow it to gently rest on the surface of the dish 4 Following instructions for the BTX generator and set the appropriate parameters 5 Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation 6
29. hieved 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 e AC waveform of 1 MHz optimal for fusion e Electroporation capabilities e A wide range of voltages from 10 V to 3000 V e Finer voltage discrimination e Capable of operating at low impedance loads of 20 Q 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 bt lt harva
30. inate 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 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 electri
31. istributor 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 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 P
32. lease 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 grounding 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 DO NOT OPERATE IN LOW IMPEDANCE Sample Load or Sample If the electroporation samples have an impedance of less than 20 Q in LV and 40 Q in HV the samples may arc and result in sample loss and potential damage to unit 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
33. m 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 7 NUMBER OF PULSES 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 8 WAIT This light illuminates when the instrument has reached the end of an operating 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 9 AUTOMATIC START Press and release to initiate an alignment and electroporation cycle according to the preset voltage pulse length and number of pulses 10 REMOTE CONTROL Optional Accessory Connect the remote control box cable so the generator can be operated from the remote control box 11 NUMBER OF REPEATS The thumbpot can be used to set the number of repeat cycles It can be set up to 9 cycles Page 14 www btxonline com ECM 2001 Electrofusion Electroporation Systems Quick Start Continued 12 MANUAL START 13 PROCESS LEDS Page 15 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 timeou
34. mm Gap 45 0113 508 Genetrode Straight 5 mm 45 0114 510 Genetrode Straight 10 mm 45 0115 512 Genetrode Bent L Shaped 5 mm 45 0116 514 Genetrode Bent L Shaped 3 mm 45 0117 516 Genetrode Bent L Shaped 1 mm 45 0118 520 Tweezertrode Electrode 7 mm diameter 45 0119 522 Tweezertrode Electrode 10 mm diameter 45 0122 542 Genepaddles 3 x 5 mm 45 0123 543 Genepaddles 5 x 7 mm 45 0124 610 Cuvette 1 mm Gap pkg 50 45 0125 620 Cuvette 2 mm Gap pkg 50 Page 39 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix C Accessories and Replacement Parts continued Catalog Number Model Description 45 0126 640 Cuvette 4 mm Gap pkg 50 45 0130 PP35 2P Petri Pulser 2 mm Gap 45 0203 515 Genetrode Genepaddle Holder Spare Parts Catalog Number Model Description 06 004009 01 Fuse T 8 A 250 V 5 x 20 mm 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 75 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 ECM 2
35. n 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 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 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 LV Mode 4 Pulse Length Dial the pulse Length on DC Pulse Length switch 5 DC Voltage Select the ele
36. ng Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 Page 44 www btxonline com ECM 2001 Electrofusion Electroporation Systems Appendix F Connecting Electrodes continued 2 Attach the Micrograbbers onto the terminal pins of the Meander Chamber slide Polarity is not important Tape the cable to the microscope stage to act as a strain relief and to avoid movement of the slide and its wires 3 Pipette one drop of cell suspension and reagents to the Microslides Meander Chamber field 4 Following instructions for the BTX generator and set the appropriate parameters 5 Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation 6 Remove the microslides meander fusion chamber carefully and prepare for the next experiment Petri Dish Electrode 1 Plug the HV cables from the Petri Dish Electrode into the voltage output of the BTX Generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to t
37. nline 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 thumbpot 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 LV mode 10 to 500 0 01 to 0 99 msec OR LV 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 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 mode 6 SET VOLTAGE 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 fro
38. ocol 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 e Email techsupport btx harvardapparatus com e Fax 508 429 5732 e Phone 1 800 272 2775 Thank you and we will do our best to assist you BTX Technical Service Group Page 48 www btxonline com HARVARD APPARATUS The Electroporation Experts 84 October Hill Road Holliston MA 01746 Phone 508 893 8999 Toll Free 800 272 2775 U S Only Fax 508 429 5732 E mail techsupport btx harvardapparatus com Web www btxonline com
39. ogeneous 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 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
40. or 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 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 ECM 2001 is changed in 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
41. rdapparatus com or your distributor A 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 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 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 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 btxo
42. resh 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 yes Fusion Did you set up the instrument right Change pre fusion treatment were cells exposed z Fusion to hypertonic solution Ve us 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 Yee 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 bt
43. t 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 Control Type Mini Din 6 pos Female Pinout Pin Signal 1 NIC 2 RTN 3 12V 4 Auto Start 5 Manual Start 6 N C 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 2 Remove the Power Cable from the wall outlet 3 Remove the Power Cable from the ECM 2001 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 38 for ordering information Install Fuse Holder Drawer refer to illustration Install the Power Cable into the ECM 2001 Plug Power Cable into wall outlet 10 Turn power switch to the ON position TA iV S
44. ther Category 8 or 9 and are currently 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 e Do Not Dispose Product with Municipal Waste e Special Collection Disposal Required ECM 2001 Electrofusion Electroporation Systems Table of Contents General Information Serial NUMbEe Taisin 2 1G EI KEE 2 Mara EE 2 3 DEEVICE ie aceewsastsnsseytarcdsnseucnsssenead senses uestevsuesachiedvesnssdtons 3 4 Repair Facilities and Parts s sessecssseseeseseeceseeseeeeeeees 4 General Safety SUMMALY s ssssesssssssssessesseeseeseess 5 6 Electrical amp Technical Specifications eessen 7 8 General Specifications sessessecsesssssssssssssssessesseess 9 Introduction IR ITT 10 Operation Getting Started s sscssssssssssssseseees 11 12 Quick Start Front Panel Control External COMMECHONS arisini Replacing Power Entry Module Fuse Operating Basics ssesssesesosssosecoseeosecosecsescsseosecsssess Advanced Operation Programming Monitoring COMMeCtiON iss etnsirnserisis kis 21 22 Experimental MethodS esssseecesesseeccossseeceesssecceossse 23 28 Appendix A Troubleshooting s esessesesecoosseeccessseeee 29 34 Appendix B Glossary of Technical Terms 35 38 Appendix C Accessories and Replacement Parts 39 40 Appendix D General Cleaning and Maintenance 4
45. ued 4 Use the thumb wheel to slide the electrodes open 5 Place the Flat Pack Chamber in between the electrodes with the aluminum coming in contact with the electrodes 6 Secure the Flat Pack Chamber in place by closing the gap with the thumb wheel The Flat Pack Chamber should be snugly placed between the electrodes however it should be loose enough that it can be pulled out without adjusting the thumb wheel again 7 Following instructions for the BTX generator set the appropriate parameters 8 Deliver the electroporation pulse s to the sample Warning Use proper eye protection during electroporation 9 Remove Flat Pack Chamber and extract the cell solution 10 Dispose of Flat Pack Chamber and prepare for the next experiment Flat Electrodes 1 Fill the sterile Flat Electrode Chamber using a syringe 2 Replace the clear plastic tip so the holes in the top align with the holes in the electrode bars 3 Push the banana plugs at the opposite end of the Flat Electrode Catalog number45 0217 into the voltage output of the BTX generator Warning Make sure the Generator is turned off prior to connecting any cables to it If using the Enhancer 3000 to monitor output connect the banana plugs into the output ports of the High Voltage Probe Use the black and red high voltage cables to connect the BTX generator to the input ports of the Enhancer 3000 4 Following instructions for the BTX generator set the appropriat
46. usion 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 BTX ECM ID 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 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 pon nn sr 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 tub
47. 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 J yes Alignment but do not align 5 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 com 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 e g Fusion amplitude alignment time GE 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 f
48. xonline 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 Ah 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 If no repeat 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 illum
49. yp 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 continued Instrument Settings Typical fusion yields should average approximately 20 with the addi tion of protease and 7 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 Voltage Time Pulse Width AC ALIGNMENT 035V 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 10 neonatal calf serum Maintain monolayer culture at 60 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
Download Pdf Manuals
Related Search