Cyto Pulse Sciences, Inc.
Contents
1. Cyto Pulse Sciences Inc Model PA 101 Electrofusion System User Manual Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 1 410 787 1890 1 410 787 1891 FAX www cytopulse com Cyto Pulse Sciences Inc makes no warranty with respect to the product except for the warranty set forth in this Users Manual on page 5 1 The LIMITED WARRANTY SET FORTH ON PAGE 5 1 IS EXCLUSIVE AND NO OTHER WARRANTY WHETHER WRITTEN OR ORAL IS EXPRESSED OR IMPLIED Cyto Pulse Sciences Inc SPECIFICALLY DISCLAIMS IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE License Agreement PA 101 Electrofusion Cyto Pulse Sciences Inc Licensor conveys to the licensee for fee paid a nonexclusive nontransferable license to use the PulseAgile hardware and software equipment for research purposes into perpetuity Cyto Pulse Sciences Inc has patent allowance and patents pending covering the PulseAgile and other process If the licensee wishes to use the hardware and software for production or commercial purposes an additional license shall be required The equipment is not approved by the FDA for use with in vitro or in vivo diagnostics or therapy The information in this manual is subject to change without notice Copyright 2000 2005 Cyto Pulse Sciences Inc PA101UMANrev 1 1 05 Price 100 00 FE Ce PA 101 User Manual Rev 1 1 05 Cyto Pulse Sciences Inc Model PA 101 Electrofusion System2. 4 Getting started Sample Protocol 4 1 Published Protocols If possible start with a published protocol and optimize parameters to suit your needs For many of the standard uses for electrofusion there are several published protocols The following list is a sample of published values Sine wave Fusion Sine wave Cells pre fusion post fusion none 0 V cm 700 V cm 5 15 sec 2 pulses 70 uS 20 50 V cm 1 5 kV cm 2 MHz 3 7 4 kV cm 10 sec 10 uS 300 V cm 750 V cm 600 Khz 3 pulses 5 10 sec 100 uS 200 V cm 2 5 kV cm 800 kHz 20 uS 250 V cm 2 25 kV cm 1 5 MHz 3 pulses 250 300 V cm 1 5 5 kV cm 3 pulses 28 35 V cm 1 4 2 kV cm 1 MHz Activation step only multiple pulses 100 V cm 2 MHz 15 30 sec 300 Vicm tapering to 0 over 1 min 250 V cm 1 5 MHz 30 sec Reference SP2 O single cells X63 mouse myeloma human mouse heteromyeloma human B cells K6H6 B5 myeloma plant protoplasts Note that all voltage values are given in volts cm This is the convention To convert the volts cm to applied voltage multiply the volts cm by the gap For example with the coaxial electrode the gap is 2 5 mm so the V cm will be multiplied by 0 25 cm to obtain the applied voltage For 200 V cm the applied voltage would be 50 volts Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 PA 101 User Manual Rev 1 1 05 4 2 Programming a protocol Similar to the elec
3. e increasing the voltage of the pre fusion sine wave e increasing the time of pre fusion sine wave application e Increasing the concentration of cells Increased pearl chain length may not always be of advantage Cell fusions of two cells only are the usual goal so a short pearl chain may be optimal Pearl chains of a range of lengths will be present in any given dielectrophoresis application Cell fusion between any two cells is a matter of probability related to the specific nature of the cells involved the electrofusion medium and the applied electric fields Because of this two cell electrofusions are possible in longer pearl chains The goal of electrofusion at least as applied to hybridoma formation is to generate the most B cell myeloma hybridomas Fusions of like cells either B cells or myeloma are not productive This is a matter of probability with approximately 50 percent of the two cell fusions being B cell myeloma hybridomas Other ratios are possible due to the difference in size of the B cells and myeloma cells Optimization of fusion pulse voltage width and number is achieved empirically Systematically varying one parameter at a time while measuring hybridoma yield is necessary to optimize fusion pulse parameters A faster experimental design is to use factorial analysis for the optimization This method allows measurement of interactions among variables and is more efficient than varying one parameter at a time
4. 4 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 PA 101 User Manual Rev 1 1 05 5 Customer Service 5 1 Limited Warranty CYTO PULSE products are warranted against defect in materials and workmanship If the customer provides notice of such a defect during warranty period CYTO PULSE at its option will either repair or replace the products which were found to be defective The limited warranty set forth above is exclusive and no other warranty whether written or oral is expressed or implied CYTO PULSE specifically disclaims implied warranties of merchantability and fitness for a particular purpose EXCEPT AS SET FORTH ABOVE CYTO PULSE MAKES NO WARRANTY WITH RESPECT TO THE PRODUCT AND IN NO EVENT REGARDLESS OF CAUSE SHALL CYTO PULSE BE LIABLE FOR INDIRECT SPECIAL OR CONSEQUENTIAL DAMAGES OR OTHER LOSSES OF ANY KIND ARISING FROM BREACH OR WARRANTY OR OTHER USES OF THIS PRODUCT CYTO PULSE S OBLIGATION TO REPAIR OR TO REPLACE TO THE EXTENT SET FORTH ABOVE CONSTITUTES THE EXCLUSIVE REMEDIES OF THE CUSTOMER FOR ANY BREACH OF WARRANTY This warranty shall not apply to products which after inspection by CYTO PULSE were found to be improperly used or to have been modified in any manner CYTO PULSE recommends that the user not open the product cabinet This limited warranty is valid for one year from the date of shipment 5 2 Customer Service If the user believes that there is a defect in the CYTO
5. Analytical Biochemistry 216 271 275 16 Hui SW Stenger DA Electrofusion of cells Hybridoma production by electrofusion and polyethylene glycol 1993 Methods in Enzymology 220 212 227 17 Kugler A et al Regression of human metastatic renal cell carcinoma after vaccination with tumor cell dendritic cell hybrids 2000 Nature Medicine 6 3 332 336 18 Schmitt JJ Zimmerman U Enhanced hybridoma production by electrofusion in strongly hypo osmolar solutions 1989 Biochem et Biophysica Acta 983 42 50 19 Neil GA Zimmerman U Electrofusion 1993 Methods in Enzymology 220 174 196 20 Collas P Fissore R Robl JM Preparation of nuclear transplant embryos by Electroporation 1993 Analytical Biochemistry 208 1 9 21 Tatham BG Gilliam KJ Trounson AO Electrofusion parameters for nuclear transfer predicted using isofusion contours produced with embryonic cells 1996 Molecular Reproduction and Development 43 306 312 2 8 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 101 User Manual Rev 1 1 05 3 PA 101 Set Up and Operation 3 1 Introduction The PA 101 is an attachment to the PA4000S PulseAgile electroporator It provides the capability of applying a low voltage megahertz frequency sine wave to the cell suspension prior to and after application of a high voltage pulse The applied sine wave serves the purpose of aligning cells in a line called a pearl chain The process is cal
6. O Box 609 Columbia MD 21045 410 787 1890 PA 101 User Manual Rev 1 1 05 In the Electrofusion Mode both pre pulse and post pulse sinusoidal AC may be added Figure 3 3 shows the components of electrofusion waveforms Pulse Amplitude Pulse Width Frequency Stop AC am DIU cinerea a Start AC amplitude w AG Duration Pre Pulse AC Wave Post Pulse AC Wave Figure 3 3 PA 101 Waveforms Settings available for the PA 101 are as follows Start 5 75 volts peak step size 5 volts Stop 5 75 volts peak step size 5 volt Frequency 0 20 to 2 MHz step size 0 2 MHz Duration 1 to 400 seconds If zero in entered in the duration no sine wave occurs Form example if zero is entered in the post sine duration there is no post sine wave delivered It is important to understand the various terms for the amplitude of the AC waveform Volts peak the amplitude from zero to the peak Volts peak to peak The amplitude from the bottom peak to the top peak The PA 101 produces a maximum amplitude of 75 volts peak or 150 volts peak to peak The screen for the Electrofusion mode is presented in Figure 3 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 3 3 PA 101 User Manual Rev 1 1 05 PA 4000 Interface Version 1 71 sinetest 5 70_pro a Figure 3 4 Set up Screen PA 101 3 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 787 1890 PA 101 User Manual Rev 1 1 05
7. PULSE product the customer should contact CYTO PULSE Customer Service through our website at www cytopulse com or phone 410 787 1890 or contact the local CYTO PULSE representative A determination if the product is still in warranty will be made If the warranty period is still in effect the user will be given an authorization number RMA to return the product If after receipt and inspection the product is found to be defective it will be replaced or repaired and returned to the customer If the product is found to have been modified or misused the user will be given a quote for repair If the warranty period has expired and the user requests repair CYTO PULSE will inspect the product and provide a written quote for repair The user must provide a purchase order number before the product will be repaired If the unit is damaged in shipment the user must recover the insured value to replace or repair from the carrier Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 5 1 PA 101 User Manual Rev 1 1 05 Blank page 5 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890
8. and other preparatory needs It is important that all tissue culture medium be washed from the cells prior to electrofusion This requires a minimum of one centrifugation plus two washes in electrofusion medium Residual ions from an incomplete wash may ruin the electrofusion For the cell washes it may be useful to wash the two cell populations separately This has two advantages One is that if there is differential cell loss during the washes compensation can be made during the mixing of the cells after the last wash For instance myeloma cells are larger than B cells and may centrifuge differently Another advantage of combining the cells after the last wash is that the two cell populations may be stained with fluorescent dyes to differentiate the two cells after fusion and to identify fused cells Stained cells must be washed in separate washes to prevent bleeding of the dye from one population to the other during the washes If the cells used in the electrofusion are immortalized tissue culture cells the process is most efficient when cells in log growth phase are used Primary cells may not divide in tissue culture and therefore may not be obtained in log phase It is also advisable to do the washes immediately prior to electrofusion Electrofusion medium is non toxic but cells will become fragile after excessive time gt 1 hour in the electrofusion buffer Buffer quality control The following should be evaluated e PH 7 3 0 1 e Os
9. related to resistance ohms by the formula l R p ve ohms where p Resistivity in onm cm o Conductivity in S cm 1 p Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 1 PA 101 User Manual Rev 1 1 05 plate spacing A plate area For a coaxial electrode the equation is In a b 2h R p ohms where p Resistivity in onm cm o Conductivity in S cm 1 p a inner electrode diameter b outer electrode diameter h electrode height Common resistivity measurements are in the range of 7 000 to 17 000 Q cm for cell fusion medium This is equivalent to a conductivity of 143 to 60 uS cm Conductivity is the reciprocal of resistivity To obtain a medium with a high resistivity or low conductivity a low ionic medium is used The resistance of the three commercially available Cyto Pulse coaxial electrodes when filled with Cyto Pulse low conductivity media CPS LCM C to the indicated volume is Cyto Pulse Electrode Units FE 25 400 FE 25 800 FE 20 1000 Volume ul 350 750 1000 Resistance ohms 2 874 1 341 617 Inner diameter mm 7 9 7 9 18 0 Outer diameter mm 13 13 22 Gap mm 2 5 2 5 2 0 Height mm 5 0 10 0 10 0 Another important property of the electrofusion medium is the osmolarity The osmolarity of electrofusion media is usually iso osmolar normal osmolarity of body fluids 290 mOs The use of hypo osmolar media has been reported 10 Hypo osmolar me
10. 5 USA 410 787 1890 PA 101 User Manual Rev 1 1 05 Table of Contents page Introduction 1 1 Electrofusion Background 2 1 2 1 Introduction 2 1 2 2 Preparation for electrofusion 2 1 2 2 1 Media 2 1 2 2 2 Cleaning and Sterilization of Electrode 2 2 2 2 3 Washes and other preparatory needs 2 3 2 3 Three step electrofusion 2 3 2 3 1 Step 1 Cell alignment 2 3 2 3 2 Step 2 Fusion Pulse 2 6 2 3 3 Step 3 Post fusion cell alignment maintenance 2 6 2 4 Post fusion cell care 2 7 2 5 Electrofusion references 2 7 aa 101 Set up and Operation 3 1 Introduction 3 1 a Connecting the PA 101 to the PA 4000 3 1 3 3 PA 101 Front panel functions 3 2 3 4 Turning unit on 3 2 3 5 Coaxial Electrode 3 2 3 6 Software 3 4 ran Started 4 1 Published protocols 4 1 1 Programming a protocol 4 2 4 3 General electrofusion method Materials 4 2 4 3 1 Electrofusion apparatus 4 2 4 3 2 Cell fusion medium 4 2 4 3 3 Complete cell growth medium 4 2 4 3 4 Electrode 4 2 4 3 5 Microscope 4 2 4 4 General electrofusion method cell preparation 4 3 4 5 General electrofusion method electrofusion 4 3 4 6 Optimizing 4 4 Customer Service 5 1 5 1 Limited Warranty 5 1 5 2 Customer Service 5 1 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 iil PA 101 User Manual Rev 1 1 05 List of Figures Non uniform Field Force Cell Alignment Clausius Mossotti Function Cell fusion system Back panel interconnections Components of electrofusion wave
11. User Manual Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 1 410 787 1890 1 410 787 1891 FAX www cytopulse com Cyto Pulse Sciences Inc makes no warranty with respect to the product except for the warranty set forth in this Users Manual on page 5 1 The LIMITED WARRANTY SET FORTH ON PAGE 5 1 IS EXCLUSIVE AND NO OTHER WARRANTY WHETHER WRITTEN OR ORAL IS EXPRESSED OR IMPLIED Cyto Pulse Sciences Inc SPECIFICALLY DISCLAIMS IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE License Agreement PA 101 Electrofusion Cyto Pulse Sciences Inc Licensor conveys to the licensee for fee paid a nonexclusive nontransferable license to use the PulseAgile hardware and software equipment for research purposes into perpetuity Cyto Pulse Sciences Inc has patent allowance and patents pending covering the PulseAgile and other process If the licensee wishes to use the hardware and software for production or commercial purposes an additional license shall be required The equipment is not approved by the FDA for use with in vitro or in vivo diagnostics or therapy The information in this manual is subject to change without notice Copyright 2000 2005 Cyto Pulse Sciences Inc PA101UMANrev 1 1 05 Price 100 00 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 i PA 101 User Manual Rev 1 1 05 Blank page ii Cyto Pulse Sciences Inc P O Box 609 Columbia MD 2104
12. d be to use a filter to exchange the medium Cells do not do well in cell fusion medium over extended periods of time Incubation beyond the 30 minutes required for fusion maturation is not recommended For hybridoma selection standard protocols using selective medium are used Exposure to selective medium may be delayed for one day to allow cells time to stabilize 2 5 Electrofusion References 1 Neil GA Zimmermann U Electrofusion Methods Enzymol 1993 220 174 96 2 Vienken J Zimmermann U An improved electrofusion technique for production of mouse hybridoma cells FEBS Lett 1985 182 278 80 3 Ohno Shosaku T Okada Y Electric Pulse induced fusion of mouse lymphoma cells Roles of divalent cations and membrane lipid domains J Membrane Biol 985 85 269 80 4 Elsheikh AS Takahashi Y Tanaka H Hishinuma M Kanagawa H Electrofusion of zona free mouse embryonic cells in electrolytes and their development in vitro Jon J Vet Res 1995 43 125 34 5 McClenaghan NH Barnett CR O Harte FP Swanston Flatt SK Ah Sing E Flatt PR Characteristics of BRIN BG5 and BRIN BG7 two novel glucose responsive insulin secreting cell lines produced by electrofusion J Endocrinol 1996 148 409 17 6 Ogura A Matsuda J Yanagimachi R Birth of normal young after electrofusion of mouse oocytes with round spermatids Proc Natl Acad Sci U S A 1994 91 7460 2 7 Willadsen SM Nuclear transplantation in sheep embryos Nature 1986 320 63 5 8 Vienken J Zi
13. dium reportedly increases electrofusion efficiency The efficiency increase occurs if cells are exposed to hypo osmolar medium then returned to iso osmolar medium The lasting effect may be due to changes in the cell cytoskeleton due to cell swelling The use of iso osmolar medium in the final wash is recommended Cyto Pulse Cytofusion medium is an optimized iso osmolar electrofusion medium designed for optimal results Cyto Pulse recommends that this medium be used for electrofusion 2 2 2 Cleaning and sterilization of the electrode before use The integrity of the electrofusion process depends upon low conductivity Any contaminants remaining in the electrode from previous electrofusion runs will hinder the 2 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 101 User Manual Rev 1 1 05 electrofusion A thorough cleaning immediately after use is required The following process should be used 1 Cleaning a Rinse the electrode well in reagent grade water b Fill the electrode with 4 sodium hydroxide and soak for 5 minutes c Rinse for 10 seconds in reagent grade water empty and repeat 10 times d Rinse in 70 Ethanol or Isopropanol e Air dry 2 Sterilization of electrode one of two processes can be used a Soak in 70 ethanol for 10 minutes or b Autoclave for 20 minutes at 15 psi This is a better method of sterilizing but it will limit the life of the electrode 2 2 3 Washes
14. ect the cable with the RCA type phono plug from the FE 10 to the Cuvette Interlock connector on the back of the PA 4000 Figure 3 2 Back Panel Interconnections Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 3 1 PA 101 User Manual Rev 1 1 05 3 3 PA 101 Front Panel Functions There are four indicators on the front panel e Power On Green LED light emitting diode This indicates that the Main Power cord is connected properly and should be on if the Main Line Power is on Interface Green LED This indicates if the DB25 Interface Cable is connected properly and should be on if the Main Line Power is on e AC ON Red LED This is illuminated when the AC sine wave is on e Load This indicator is a series of 10 separate LEDs It indicates the relative resistance of the load and therefore the amount of heat that is being generated in the coaxial electrode In normal operation segments up to five may be illuminated depending on the volume of buffer cells in the electrode If five segments are lit this is approximately a 400 Q load with a 75 volt peak applied voltage at 1 MHz frequency If there are more than five segments illuminated significant heat is being generated indicating too many ions in the solution 3 4 Turning the unit on After the set up described in Chapter 4 of the PA 4000 manual and section 3 2 of this manual is complete the unit is ready to be used The following sequence of
15. egative it determines the direction of the force on the cell and thus the direction it will move If the properties of the medium are the same as the cytoplasm then K 0 and no force is applied Thus the properties of the medium are very important See Figure 2 3 The r in r is the radius of the cell Thus the force on the cell increases as the cube of the radius Large cells have much more force applied than smaller cells There is also a countervailing force the drag on the cell by the medium The larger the cell the larger the drag VE describes the non uniformity of the electric field in volts mm If the electric field is constant like in a cuvette with parallel plates then this term is zero and no force is applied to the cells The geometry of the electrode defines this value Also note that since the electric field term is squared the polarity of the electrodes makes no difference The direction of movement is dependent on the electrode geometry Clausius Mossotti Function cell radius 7 um 1 000 0 800 0 600 0 400 0 200 K 0 000 0 200 0 400 0 600 10 uS cm DOK 100 uS em 1 000 0 001 0 01 0 1 1 10 100 1000 Frequency Mhz Figure 2 3 Clausius Mossotti Function Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 5 PA 101 User Manual Rev 1 1 05 The Cyto Pulse cell fusion coaxial electrode has been optimized to convert the PA 101 sine wave voltage to an efficient no
16. events needs to be completed e Plug the Line Mains cord into the PA 4000 e Turn on the Main Line Switch on the front of the PA 4000 e Check the Power On and HV Off green LEDs are illuminated on the front of the PA 4000 e Check the Power On Green LED and Interface Green LED are illuminated on the PA 101 3 5 Coaxial Electrode The Pulse high voltage parameter settings are similar to the PA 4000 Electroporation mode There is only one voltage range 5 to 800 volts The intensity of the AC filed and pulse field is shown in Section 2 3 6 Software Details of software installation and operation of the PA 4000 are described in Chapter 4 of the PA 4000 manual The software that operates the PA 101 is included in the PA 4000 software file When the PA 101 Interface cable is connected the software is activated e Open the PulseAgile application software e Inthe Options Connected area at the upper right the PA 101 block is now checked This indicates the interface between the PA 4000 and the PA 101 is working properly e Either set up or open an electrofusion protocol If the user has developed protocols previously just open the protocol folder and click on the desired protocol If this is the first time then a new protocol must be programmed Click Electrofusion on the upper left of the screen Then click ADD at the right of the screen and enter the parameters values and save the protocol 3 2 Cyto Pulse Sciences Inc P
17. forms Electrofusion mode screen Example of cell alignment with K562 cells Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 101 User Manual Rev 1 1 05 Caution Notice This instrument contains a high voltage power supply adjustable beyond 1 000 volts such voltage can be lethal The user must read this manual carefully before the instrument is placed into operation Removing the cover may void the warranty Do not connect or disconnect the high voltage cable with the high voltage enabled To connect or disconnect the cable turn line power off and unplug line cord Do not open the cuvette holder while the high voltage is on If a problem occurs during a run push the STOP RESET button on the front panel If there are any questions about the operation of this instrument call Cyto Pulse Customer service Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 v PA 101 User Manual Rev 1 1 05 vi Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 101 User Manual Rev 1 1 05 1 Introduction Electrofusion has many uses in the fields of cell biology medicine and microbiology The PA101 add on to the PA 4000 computer controlled electroporator provides cell alignment capabilities in addition to the versatile capabilities of the PA 4000 All parameters are computer controlled to give the operator maximum flexibility in performing cell fu
18. he direction of the field Electric fields 2 3 times threshold can be used This should be optimized for the cells used Multiple fusion pulses may be more efficient than a single pulse The table below gives the approximate electric field intensity for a pulse of 800 volt in the Cyto Pulse electrodes Part No Gap Inner Mid Outer mm V cm V cm V cm FE 25 400 2 5 4087 3107 2496 FE 25 800 2 5 4087 3107 2496 FE 20 1000 2 0 4430 3990 3624 2 6 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 101 User Manual Rev 1 1 05 2 3 3 Step 3 Post fusion cell alignment maintenance The last step in the electrofusion process is post fusion alignment maintenance Electrofusion is a process that continues to occur over some time after the fusion pulse is applied Re applying dielectrophoresis at a lower AC voltage the cell are already aligned after the fusion pulse allows maturation of the fusion process by holding cells in optimal alignment and contact 2 4 Post fusion cell care Cell fusion continues to occur over 10 to 30 minutes after electrofusion The cells are very fragile at this point and should not be disturbed Cells should sit in the cell fusion electrode for 30 minutes At the end of the 30 minute time the cells can be washed by centrifugation Centrifugation is the most common method for exchanging cell fusion medium for tissue culture medium An alternate gentler method woul
19. iform electric fields Merely applying a uniform electric field will not move a cell because the net charge of the cell is zero Thus from the definition of electric field there is no force applied Force Electric Field charge However a non uniform field moves the positive ions inside each cell to one side and the negative ions to the opposite side producing a dipole Once the dipole is induced a net force is exerted on the cell because the intensity of the field is greater on one side than the other The movement of cells in one direction causes the cells to concentrate in an area Since the cells are now dipoles the negative side of one cell will attract the positive side of another Figure 2 1 Non Uniform Field Force Figure 2 2 Cell Alignment cell overcoming the negative surface charge This pearl chain process is illustrated below The equation that predicts the forces applied by this very complex process is Fy MmAK r VE This equation says three very important things 2 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 1 PA 101 User Manual Rev 1 1 05 K is called the Clausius Mossotti equation and has a maximum value of 1 and a minimum value of 1 2 The value is determined by the frequency of the voltage waveform the dielectric constant and conductivity of the cell cytoplasm the dielectric constant and conductivity of the medium Since this parameter can be positive or n
20. las are described in Chapter 2 4 3 3 Complete cell growth medium T he growth medium used will be cell type dependent One cell growth medium is as follows e Dulbeco s Modified Essential Medium 500 ml e Heat inactivated fetal bovine serum 75 ml e L glutamine 100X 5 ml e Antibiotic mixture 100X 5 ml 4 3 4 Electrode Coaxial electrode 2 5 mm gap 100 400 ul capacity 4 3 5 Microscope An inverted microscope with long focal length objectives is needed 4 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 PA 101 User Manual Rev 1 1 05 4 4 General Electrofusion Method Coaxial Electrode Cell Preparation e If desired stain cells using fluorescent labels according to the cell labeling protocol e Count the cells in each population Prepare an iso osmolar low conductance fusion medium or use Cyto Pulse commercial medium e Wash the cells 3 times in the new medium A thorough wash is necessary Centrifuge cells at 400 X g for 10 minutes Pour off the supernatant Re suspend the pellet with a gentle flick and adding 10 ml iso osmolar or hypo osmolar fusion medium Centrifuge cells at 400 X g for 10 minutes Pour off the supernatant Re suspend the pellet with a gentle flick and adding 10 ml iso osmolar fusion medium Centrifuge cells at 400 X g for 10 minutes e Re suspend the cells at 1 to 10 million cells ml in iso osmolar fusion medium after the last centrifugation Recount the ce
21. led dielectrophoresis and is useful for placing cells into proper contact for electrofusion The dielectrophoresis unit is designed to work with a high resistance load Do not use highly ionic buffers in the devices for this unit If ionic buffers are used two things will happen First the sine wave will not effectively induce pearl chain formation Second and most important a high amount of heat will be generated killing all of the cells Figure 3 1 Cell Fusion System PA 4000 PA 101 3 2 Connecting the PA 101 to the PA 4000 Follow the instructions in Chapter 4 of the PA 4000 manual to set up the PA 4000 The PA 101 should be in place on top of the PA 4000 There are five connections on the back of the PA 101 The Line Mains Switch must be off and the cord unplugged from the PA 4000 e Attach the option interconnect cable to the DB25 jacks on the back of the PA 101 and the PA 4000 The option interface cable is a 25 wire ribbon with a DB25 plug on each end e Plug the Line Mains cord from the PA 101 into the Hardware Option Power outlet on the back of the PA 4000 e Connect the high voltage cable This cord delivers the high voltage pulse generated by the PA4000 to the PA 101 It is a coaxial cable with MHV connectors on each end DO NOT ATTEMPT TO USE A CABLE WITH BNC CONNECTORS e Connect the high voltage cable from the FE 10 Fusion Plate Holder to the AC Pulse OUT SHV jack on the back of the PA 101 e Conn
22. lls after the last centrifugation to make sure that cells were not lost in the washes e Mix cell populations together in a pre determined ratio 1 1 to 1 5 On the second centrifugation of the wash a hypo osmolar medium may be used to increase cell fusion The hypo osmolar medium is made by diluting the iso osmolar medium with three parts sterile distilled water and one part iso osmolar medium The cells should sit at room temperature for 5 minutes in the hypo osmolar medium before the centrifugation 4 5 General Electrofusion Method Coaxial Electrode Electrofusion K562 cell example e Add 400 ul cell suspension to the coaxial fusion electrode e Program the PA 4000 for a pre fusion sine wave dielectrophoresis AC of 35 volts beginning and ending voltage at a frequency of 1 2 MHz for 30 seconds e Program the PA 4000 for 2 fusion pulses at 500 volts 2 KV cm and 50 us duration e Program the PA 101 for a post fusion sine wave dielectrophoresis AC of 25 volts beginning to 5 volts ending ramp down at a frequency of 1 2 MHz for 60 seconds e Place the electrode in the holder see use of electrofusion holder e Turn the high voltage on and start the process e After the electrofusion is finished remove the coaxial electrode and place it ina covered moist dish at 37 C for 30 minutes to allow fusion maturation e Move cells from the fusion electrode to a 15 ml conical tube e Rinse remaining cells from the fusion electrode to the 15
23. ml conical tube e Wash the cells twice in 10 ml complete growth medium Add 8 10 ml complete medium to the conical tube Centrifuge cells at 400 X g for 10 minutes Pour off the supernatant Re suspend the pellet with a gentle flick and add 10 ml complete medium Centrifuge cells at 400 X g for 10 minutes Pour off the supernatant Re suspend the pellet with a gentle flick e Re suspend the cells in the required amount of complete growth medium and incubate at 37 C Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 4 3 PA 101 User Manual Rev 1 1 05 Note This is one example The parameters listed in this protocol will need to be optimized for your cells gt gt r ata f F lt F AF gt jod ci so z i ence Re P Pr i pe Pia bee vi ber Na Pur pr er AGS Sa de rm ond j oi sri 2 rs a Ape Sy RPN Cheri Deel bee DE es Dy x of vred p x YLE I 1 La HA AVR Ai Pots of 5 z ne St Eg rett 4 a y i 3g b S gt 4 ve s lel mi DN N i iu gt A bed be i M rm t ya ye i 000 Sth SD ATK fF s E md 14 4 ris 5 re hes AAT 1 HUL ADA Ly w i sA 4 ba WA ei gt i gt mst SED he TT ka NE Figure 4 1 Example of cell alignment with K562 cells 4 6 Optimizing Dielectrophoresis is an observable event using a microscope with Cyto Pulse coaxial electrodes Pearl chain length can be increased by
24. mmermann U Fouchard M Zagury D Electrofusion of myeloma cells on the single cell level Fusion under sterile conditions without proteolytic enzyme treatment FEBS Lett 1983 163 54 6 9 Wojchowski DM Sytkowski AJ Hybridoma production by simplified avidin mediated electrofusion J Immunol Methods 1986 90 173 7 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 7 PA 101 User Manual Rev 1 1 05 10 Zimmermann U Gessner P Schnettler R Perkins S Foung SK Efficient hybridization of mouse human cell lines by means of hypo osmolar electrofusion J Immunol Methods 1990 134 43 50 11 Foung S Perkins S Kafadar K Gessner P Zimmermann U Development of microfusion techniques to generate human hybridomas J Immunol Methods 1990 134 35 42 12 Eigel L Oelmuller R Koop HU Transfer of defined numbers of chloroplasts into albino protoplasts using an improved subprotoplast protoplast microfusion procedure transfer of only two chloroplasts leads to variegated progeny Mol Gen Genet 1991 227 446 51 13 Steenbakkers PG van Meel FC Olijve W A new approach to the generation of human or murine antibody producing hybridomas J Immunol Methods 1992 152 69 77 14 Grobner U Berg H Polylysine supports electrofusion Bioelectrochemistry and Bioenergetics 1992 39 181 184 15 Jaroszeski Mark J Gilbert Richard Heller Detection and Quantitation of Cell Cell Electrofusion Products by Flow Cytometry 1994
25. molarity 285 295 mOs e Conductivity lt 100 microsiemens cm e Sterility e Endotoxin level 2 2 4 Mixing the Cells Prior to putting the cells into the electrofusion electrode the two cell populations need to be mixed The ratio of cells in one population to the other cell population should be Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 3 PA 101 User Manual Rev 1 1 05 approximately one to one However uneven ratios may be needed if hetero fusions are not as high as expected i e 2 of the fusions The concentration of cells during electrofusion needs to be consistent from time to time because the length of pearl chain is cell concentration dependent Cell concentrations from 1 million to 10 million cells per ml are generally used 2 3 Three step electrofusion 2 3 1 Step 1 Cell alignment The first process in any cell fusion system is to bring the cells into contact This can be done by one of many methods Chemical methods such as Avidin biotin bridging specifically selects which two cells are to come into contact Chemical methods require more manipulation than other methods but can be useful in certain circumstances Physical methods such as centrifugation can bring cells into contact prior to or after the fusion pulse In any case sufficient force must be applied to each cell to overcome the negative surface charge 2 3 1 1 Non Uniform Electric Field The Cyto Pulse PA 101 uses non un
26. n uniform alternating field for cell alignment It also converts the PA 4000 high voltage pulse s to a DC pulse electric field for cell fusion The alignment process using dielectrophoresis is a function of e The time that the AC wave is on e The intensity of the AC electric field e The frequency of the AC electric field e The non uniformity of the AC electric field The first three parameters are selected using the PA 4000 interface software and the last parameter is a property of the coaxial electrode The following chart shows the approximate intensity of the AC field for the three Cyto Pulse electrodes at a set ac voltage of 75 volts peak The field is non uniform the values below are given at the inner electrode mid way and at the outer electrode Part No Gap Inner Mid Outer mm V cm V cm V cm FE 25 400 2 5 179 135 109 FE 25 800 2 5 179 135 109 FE 20 1000 2 0 415 373 340 2 3 2 Step 2 Fusion Pulse The second step in electrofusion is to apply one or more high voltage pulses to the cells inducing membrane fusion The voltage required must be above a threshold to induce membrane breakdown and below a maximum voltage causing cell death Threshold voltage is approximately 0 5 1 5 volts across the cell membrane 1 The voltage across a cell is approximately equal to V 1 5 r E cosd volts where r cell radius in cm E electric field intensity in v cm angle of the membrane in relation to t
27. on 2 2 1 Medium The first consideration is the fusion medium Media of high quality must be used or the electrofusion will fail One example of a poor medium would be one containing excessive ions due to the addition of inorganic acids or bases in order to adjust the pH The presence of the ions will increase conductivity enough to result in excessive heat that will kill the cells Cyto Pulse Cell Fusion Medium is prepared using GLP manufacturing standards It is tested for pH endotoxin conductivity sterility and other quality control parameters For consistent results we suggest using this medium for your cell fusions The most common reason for electrofusion failure is the use of poor quality medium Cells need to be in a high resistance medium for two reasons One is that the cell alignment step dielectrophoresis is dependent upon there being a difference between the resistivity of the cell cytoplasm and the resistivity of the medium outside a cell Second the high current generated in a highly conductive media will cause excessive heating The heating will at first cause convection currents and disrupt pearl chains If the heating persists the temperature of the medium will rise to critical levels and kill the cells We use resistivity conductivity to describe the resistance of the medium because resistivity takes into account the volume of liquid in an electrode Remember that for a parallel plate electrode resistivity p is
28. sion protocols This manual is designed to help you get the most benefit from using PulseAgile electrofusion It contains information on how to operate the electroporator safety tips and information useful for successfully performing your electrofusions Note The PA 4000 with the PA 101 contains a high voltage power supply and was designed with safety features to protect the user and the equipment If used properly the PA 4000 with the PA 101 is a safe and reliable instrument Chapter 3 explains some important concepts related to operator safety in addition to concepts needed for accurate use of the instrument Chapter 3 must be read before setting up this instrument Our goal is the safe and productive use of the PA 4000 with the PA 101 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 1 1 PA 101 User Manual Rev 1 1 05 Blank Page 1 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 101 User Manual Rev 1 1 05 2 Electrofusion Tutorial 2 1 Introduction Electrofusion is a multi step process consisting of the following phases e Preparation of cells for electrofusion e Three step electrofusion Pre fusion dielectrophoresis to align cells High voltage pulse to induce fusion Post fusion dielectrophoresis to maintain alignment for maturity e Care of cells after fusion Each of these phases is critical for a successful outcome 2 2 Preparation for electrofusi
29. troporation protocols the concept of pulse groups is important Pulse groups are modified by selecting the group modifying the parameters as desired then clicking REPLACE Note that changes will not be accepted into the protocol until the REPLACE button has been selected Only one pulse group is used with electrofusion For electrofusion a pulse group is one pre sine setting one high voltage pulse and one post sine setting For electrofusion only one pulse group can be selected The number of pulses in the pulses selection window means a repeat of the high voltage pulses only For example to program the following protocol would require the settings in the table below e a1 2 MHZ pre sine wave with a ramp of 5 to 60 volts over 20 seconds e 5 pulses of 300 volts and 100 us duration and 0 125 second interval e and a 1 2 MHZ post sine wave with a ramp of 20 to 5 volts over 30 seconds Group number of Parameter Pre sine High Voltage Post sine Interval pulses Pulse Group 1 Volts Beginning 0 125 s Number 5 0 100 ms FrequencyMHz 12 12 Volts Ending 60 1 5 4 3 General Electrofusion Method Coaxial Electrode Materials 4 3 1 Electrofusion apparatus Cyto Pulse Sciences Inc PA 4000 with PA 101 dielectrophoresis accessory 4 3 2 Cell fusion medium Use Cyto Pulse Low Conductivity cell fusion medium for best results Poor quality medium is the most common reason for electrofusion failure Cell fusion medium formu
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