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Model PA-3000 Rectangular Wave Electroporation System

1. 0 998775434 Figure B 3 Typical Pulse Voltage as a Percent of Power Supply Voltage Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 B 5 PA 3000 Users Manual B 3 Using External Equipment to Measure Pulse Voltage and Current The most accurate method to measure pulse voltage and pulse current is with a high voltage oscilloscope probe and an external current transformer High Voltage Probe Most oscilloscope manufacturers offer a high voltage probe One example is the Tektronix TEK5100 To use the probe connect the probe tip to the high voltage side of the external load such as cuvette contact The ground side must be connected to the System Ground Screw on the back panel not the other side of the external load If connected to the other side of the external load the internal current monitor circuit will distort the current measurement Caution when connecting to the high voltage side care must be taken so it is not possible to come in contact with any high voltage while the system is operating Current Transformer The most accurate and safest current measurement is with a torroidal current transformer This is a coil through which the low potential side of the External Load current is passed The current through the return lead induces a voltage in the transformer which is in turn measured A Pearson Model 411 is recommended for this type of measurement Contact Pearson Electronics Palo Alto
2. C and D pore diameter and total pore area become too large for the cell to repair by any spontaneous or biological process Therefore the cell is irreversibly damaged To prevent this damage pulse protocols are empirically developed to be at some point above threshold and below lethality Figure 2 1 Electropores Total P Since the mechanism of electroporation is not well understood the development of protocols for a particular application to a previously uncharacterized cell or tissue have usually been achieved by empirically adjusting pulse parameters such as amplitude duration number and inter pulse interval ore Area Pulse Amplitude x Pulse Duration Initial Pulse A B D C Figure 2 2 Pore Area Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 1 PA 3000 User Manual Although early research on electropore mediated transport across membranes assumed that simple thermal motion i e diffusion propelled molecules through electropores research in the late 1980s and early 1990s began to reveal that movement of molecules through electropores depends on other experimental conditions and electrical pulse parameters in a way that indicates that other processes are involved These reports show that certain experimental conditions and parameters of electrical pulses may be capable of causing many more molecules to move per unit time than simple diffusion For example
3. Part No DC 100 B 1 How to Use the Internal Monitors The PA 3000 and PA 4000 have internal pulse voltage and current monitors The monitors are available via BNC connectors on the back panel of the unit These monitors provide a signal that is a scaled down replica of the actual pulse voltage and pulse current The monitor signal must be viewed with an oscilloscope The monitors must operate into a 50 ohm load to provide a properly calibrated signal This may be accomplished by selecting the 50 ohm input impedance option on those oscilloscopes that have that option or by using an external feed through 50 ohm coaxial termination A kit containing three 1 meter coaxial cables and three 50 ohm attenuators may be purchased from Cyto Pulse Part No Cable 50 Three connections must be made to use the monitors Connection 1 External Trigger this BNC connector is at the top right corner on the back panel A coaxial cable is connected between this connector and the oscilloscope external trigger input The signal is identical to the low voltage pulse that drives the high voltage switch This signal has the same width and interval as the high voltage pulse but is always the same voltage The level of this trigger pulse is about 1 5 volts into 50 ohms When used in this manner the scope will be triggered independent of the pulse voltage or pulse current amplitude A trigger level on the oscilloscope of 1 0 volt is recommended Connection 2 Pulse Vol
4. intensities high voltage pulse generators have adjustable pulse amplitudes from tens of volts to over 1000 volts Figure 2 4 presents the electric field intensity for standard cuvettes and applied pulse voltages D Voltage E E Voltage D Spacing Plate Area Figure 2 3 Electric Field The concept of resistance is also very important in this process From basic physics Ohm s Law states Current voltage resistance Current is the quantity of electrons flowing per second Resistance omega or load is the hindrance to that flow measured in ohms at the applied voltage Current is similar to water flowing in a pipe A smaller diameter pipe allows fewer water molecules per second to flow In this case water pressure is analogous to voltage In biology the solution in which the cells are contained will determine the sample s electrical resistance Solutions such as phosphate buffered saline PBS are very ionic and will conduct a large amount of current Distilled water DW and solutions containing sucrose in distilled water are not ionic and will conduct a small amount of current When discussing the conducting properties of material or solutions a common parameter used is resistivity represented by the Greek symbol rho This is given in ohm cm and is related to resistance by the formula 0 2000 4000 6000 8000 10000 12000 14000 0 200 400 60
5. 581 pp Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 2 14 PA 3000 User Manual Chapter 3 PA 3000 Overview This chapter describes the various PA 3000 system configurations accessories and important concepts on the use of the systems and operating capability 3 1 Overview This section describes the basic system configurations for the PA 3000 Available accessories are presented in Figure 3 1 Cuvettes Type Max Field Space Area Volume Part No Standard 2 7 kV cm 4 mm 2 cm x 1 cm 800 L CUV 04 Standard 5 5 kV cm 2 mm 1 cm x 1 cm 200 L CUV 02 Standard 11 0 kV cm 1 mm 1 cm x 1cm 100 L CUV 01 Chambers Electrodes Standard Cuvette Holder CE 20 Figure 3 1 Accessories The basic system is controlled by an internal digital control system This system performs three PulseAgile functions independent control of amplitude pulse width and pulse interval The Control Assembly generates the pulses used to drive the high voltage switch and the control voltage for the high voltage power supply The High Voltage Assembly is where the high voltage pulses are generated and the pulse voltage and current monitors are located The configuration provides a connection for observing the pulse amplitude and pulse current if an oscilloscope is available The voltage output is connected to the Standard Cuvette Holder The Standard Cuvette Holder also has an interlock circuit built in An
6. O Box 609 Columbia MD 21045 410 715 0990 7 1 PA 3000 Users Manual Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 7 2 PA 3000 Users Manual Appendix A Pulse Specifications Power Supply Voltage 20 to 1100 volts Step Size 5 volts Set Accuracy 5 5 volts Maximum Average Power gt 50 watts Pulse Amplitude Pulse Amplitude at 10 ohm load 20 to 925 volts at 20 ohm load 20 to 1000 volts at 100 ohm load 20 to 1050 volts at 1000 ohm load 20 to 1095 volts Pulse to Pulse Variation lt 5 Droop lt 5 at 20 ohms 100 s lt 5 at 100 ohms 2000 s Leakage at 1 000 volts lt 0 2 volts rms at no load lt 0 1 volt rms at 10 ohm load Pulse Width and Interval Pulse Width at 10 ohm load 1 s to 30 s at Int 0 13 second 1 s to 260 s at Int 1 00 second at 20 ohms load 1 s to 60 s at Int 0 13 second 1 s to 490 s at Int 1 00 second at 100 ohm load 1 s to 280 s at Int 0 13 second 1 s to 2000 s at Int 1 00 second Pulse Width Step Size 1 s Pulse Interval 0 13 second to 400 seconds Pulse Interval Step Size 0 01 second 10 milliseconds Groups Number of Groups 9 Number of Pulses per Group 99 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 A 1 PA 3000 Users Manual Monitors High Voltage Power Supply Meter 5 Load Ohm Meter 20 Pulse A
7. Optional this connector is available for the researcher who wishes to observe measure the actual pulse amplitude using an oscilloscope It is not required for operation of the unit This monitor was calibrated into a 50 ohm load and 50 ohm cable A 50 ohm terminating impedance must be used A 50 ohm cable pair with terminations may be ordered from Cyto Pulse The pulse voltage is calculated by Pulse Voltage P VMON x 200 volts P IMON Pulse Current Monitor Optional this connector is available for the researcher who wishes to observe measure the actual pulse current using an oscilloscope It is not required for operation of the unit This connector is also used by the scope option This monitor was calibrated into a 50 ohm load and 50 ohm A 50 ohm terminating impedance must be used A 50 ohm cable pair with terminations may be ordered from Cyto Pulse The pulse current is given by Pulse Current P IMON x 20 amps Sync Trigger Out Optional this connector is available for the researcher who wishes to observe measure the actual pulse signals using an oscilloscope It is connected to an oscilloscope trigger input to synchronize the pulse signal The trigger signal precedes the pulse amplitude or current signals by a few hundred nanoseconds Connection to the trigger connector is not required for operation of the unit This connector is also used by the scope option A 50 ohm cable and terminating impedance should be used A trigger level
8. of the set voltage Therefore the actual set voltage needs to be 600 0 9 667 volts 6 4 References 1 Zerbib D Amalrick F Teissie J 1985 Electric field mediated transformation Isolation and characterization of a TK subclone Biochem Biophys Res Commun 129 611 2 Bartoletti D C Harrison G I and Weaver J C 1989 The number of molecules taken up by electroporated cells quantitative determination FEBS Letters 256 4 10 3 Wolf H Rols M P Boldt E Neumann E Tiessie J 1994 Control by pulse parameters of electric field mediated gene transfer in mammalian cells Biophys J 66 524 531 4 Mir L M Banoun H Paoletti C 1988 Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization direct access to the cytosol Exp Cell Res 175 15 25 5 Serpersu E H Kinosita K Tsong T Y 1985 Reversible and irreversible modification of erythrocyte membrane permeability by electric field Biochem Biophys Acta 812 779 6 Liang H Purcker W J Stenger D A Kubiniec R T Hiu S W 1988 Uptake of fluorescent labeled dextrans by10T fibroblasts following permeation by rectangular and exponential decay electric field pulses BioTechniques 6 550 7 Press F Quilet A Mir L Marchio Fournigalt C Fuenteun J Fradelizi D 1988 An improved electro transfection method using square shaped electric impulsions Biochem Biophys Res Commun 1
9. other commercial units In addition there is a subset of the PulseAgile protocols available on the PA 4000 advanced system Cyto Pulse PulseAgile technology gives research scientists and medical scientists the tools needed for demanding new uses In addition the standard rectangular wave protocols commonly in use PulseAgile electroporation was developed to give the operator maximum flexibility in protocol design and execution Protocols can be optimized to give the best cell viability the highest transfection efficiency or the best electroporation efficiency This manual is designed to help you get the most benefit from using the PA 3000 electroporator It contains information on how to operate the electroporator safety tips some important physical concepts hints on how to adapt other protocols to PulseAgile and hints on developing your own protocols Note The PA 3000 contains a high voltage power supply and was designed with safety features to protect the user and the equipment If used properly the PA 3000 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 safe and productive use of the PA 3000 This product shall only be used in a manner specified by the manufacturer Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 71
10. pass from one side of the membrane to the other As Figure 2 1 shows the electropores are located primarily on the surfaces of cells that are closest to the electrodes If the electric field pulse has the proper parameters then the electroporated cells can recover the electropores reseal spontaneously and cells will continue to grow and express their genetic material E A B The use of electroporation became very popular through the 1980s because it was found to be an exceptionally practical way to place drugs genetic material e g DNA or other molecules into cells In the late 1980s scientists began to use electroporation protocols with multi cellular tissue as well as cell suspensions Though cell to cell biological variability causes some cells to be more sensitive to electroporation than other cells pore formation number and effective diameter is generally a function of the product of the pulse amplitude and the pulse duration Figure 2 2 In order for pores to form this product has to be above a threshold In Figure 2 2 lines A and B identify thresholds where pore formation begins Additionally pore number and effective pore diameter increase with the product of pulse amplitude and pulse duration Although other factors are involved this threshold is now understood to be largely dependent on the reciprocal of cell size If the upper limit threshold is reached lines
11. reduction of sodium in the medium is an example 2 5 3 Reporter Molecules Electroporation protocol development is much easier if a reporter molecule is available to readily assess the status of electroporation efficiency A partial list of available material follows DNA with appropriate promoters lac Z B galactosidase green fluorescent protein Chloramphenicol acetyltransferase Luciferase antibiotic resistance Non DNA FITC labeled dextrans Calcein Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 2 10 PA 3000 User Manual propidium iodide trypan blue The choice of reporter molecule is based upon 1 the similarity in composition and size of the reporter molecule to the molecule of interest and 2 the ability to assay for the reporter molecule For example it is a simple matter to screen for antibiotic resistance in bacteria that have been transfected with a plasmid containing an antibiotic resistance gene Similarly if a fluorescent microscope or a flow cytometer is available the green fluorescent protein gene under the control of a constitutive mammalian expression promoter makes an ideal reporter gene The fluorescent labeled dextrans are available in several molecular weights Proteins can be directly labeled with fluorescein Keep in mind that it is much harder to detect fluorescein labeled dextrans or proteins than it is to detect gene products because of the amplification inh
12. referring to Figure 2 1 there is good evidence that molecular flow is in the direction of the arrow A Dimitrov and Sowers 1990 However there is also good evidence that DNA movement is in the direction of the arrow B Sukharev et al 1992 This implies that electroporation has a polarity dependence Although this apparent contradiction will have to be resolved by future basic research it clearly shows that movement of molecules during electroporation is active rather than passive An additional important consideration is heat generation during electroporation During the electroporation pulse the electric field causes electrical current to flow through the cell suspension or tissue Biologically relevant buffers for cells culture medium and fluid in extra cellular space in tissues contain ions at concentrations high enough to cause high electric currents to flow These currents can lead to dramatic heating that is biologically unacceptable This is explained in more detail in the tutorial on Equipment Principles of physics suggest that the early part of an exponentially decaying pulse does most of the membrane porating but the late part continues to heat the medium as well as molecular movement One way to minimize heating is to use relatively high amplitude short duration rectangular wave pulse instead of an exponentially decaying pulse If multiple pulses are used second and subsequent pulses may be sho
13. reservoir capacitors When the high voltage is turned on this capacitor is charged to an initial voltage While a pulse is on the capacitor is connected to the load and the electrons in the capacitor are drained off just like water running out of a reservoir The longer the pulse the more electrons run out and the voltage decreases reservoir level drops just as in an exponential discharge pulser In rectangular wave pulsers the maximum pulse width is usually defined at the point that the voltage decreases to 95 of the initial voltage level The pulse width is determined by the size of the internal reservoir capacitor and the load resistance Thus when using highly ionic loads the electrons are depleted faster Caution is required in setting pulse widths in these situations The value of the reservoir capacitor in the PA 3000 is 156 F Droop is the voltage decrease from the start of the pulse to the end of the pulse Droop is calculated by Droop Pulse Width 273 F x Load R If the load is 10 ohms then the droop will be 5 in 78 microseconds 3 2 5 Maximum Pulse Width Multiple Pulse Protocols Maximum Pulse Width in multiple pulse protocols is a function of pulse droop and capacitor re charge rate In order to maintain consistent pulse voltage during multiple pulse protocols pulse width must be no wider than that shown in figure 3 4 Between pulses in multiple pulse protocols the power supply needs time to bring the capacitor vo
14. sec gt gt gt CHARGING lt lt V 225 of 600 v After the correct voltage is reached the following screen appears A non adjustable window of 90 seconds is given in which to start the running of the protocol If after the 90 seconds elapses the protocol is not started the system shuts down the high voltage Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 6 PA 3000 Users Manual Ch 5 gt gt gt PUSH lt GO gt NOW lt lt lt lt V 600 Off In 80 Step 15 Check the Fault conditions and Status Check the Fault and Status LEDs to see if the system is ready to go None of the fault LED s should be illuminated Fault Conditions Cuvette External Internal Open The power light should be green and the HVON and Off Zero lights should be red After pulsing is initiated the pulse light will flash red during each pulse Power Ready HVON Off Zero Pulse Status Step 16 Start the protocol Push the Go button to run the protocol Step 17 Protocol completion gt gt CALCULATED LOAD lt lt _ R 100 OHMS 98 Cyto pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 7 PA 3000 Users Manual Ch 5 When the protocol is complete a screen will appear that provides an estimate of the load presented by the cuvette and an estimate of the actual pulse amplitude see Sections 3 2 2 and 3 2 3 This load estimate is used by the microprocessor
15. the high voltage pulse electrodes The electrodes can only apply voltage to the cuvettes while the handle is pushed all the way in Cuvette Holder Interlock and Cable If the Cuvette Holder handle is withdrawn from the plastic shield an interlock is opened and the high voltage can not be enabled The interlock is via a second cable that must be connected for the system to operate If the handle is withdrawn a red Light Emitting Diode LED will be illuminated on the front panel and a message will appear in the LCD display Pre Pulse Load Estimator As described in section 3 2 4 a load estimator circuit is used to determine the resistance of the load before the high voltage is turned on If the value of resistance is too low below 8 ohms the PA 3000 LCD display will display a Fault error message and will not turn on If this happens there is a problem with the conductivity of the cuvette solution This problem will need to be fixed before the system will operate External Fault Detection There is a second load current detector in the system This is used primarily to detect excessive load current while the system is generating high voltage pulses For example if the pre pulse Load Estimate is above 8 ohms the high voltage will be enabled However if there is an arc in the cuvette during a pulse there will be excessive current in that cuvette This will be detected and the unit will shut down within 2 microseconds A red LED on the front pane
16. tools to design and implement optimal electroporation protocols The rest of this manual is dedicated to the description and use of PulseAgile electroporation It is goal is to provide you with the ability to get the best use of this new technology 2 3 Using PulseAgile Protocols The simplest way to start using PulseAgile protocols is to begin with published parameters for the cell type with which you are working Until PulseAgile protocol optimization is done standard published procedures and parameters can be used Both the PA 3000 and PA 4000 can readily deliver single pulses or pulse trains according to standard published specifications However optimization may be desirable in certain circumstances when cells are difficult to replace or when high yield or viability is needed PulseAgile electroporation protocols give you the flexibility to achieve your goals Optimization of an electroporation protocol is an empirical process but there are some principles that can be used to narrow the search for an ideal protocol For instance there are at least two and probably more mechanisms that have been proposed for movement of DNA into cells during transfection They are electrophoresis 1 2 and electroosmosis 4 Thermodiffusion and osmotic flow of medium also have been proposed as transport mechanisms but there is little evidence that they play more than a minor role 3 For any of these mechanisms to work the first pulse mus
17. 0 800 1000 Pulse Amplitude volts Electric Field in Cuvette v cm D 10 mm D 4 mm D 2 mm D 1 mm D 0 75 mm Figure 2 4 E Field vs Cuvette Spacing resistance D A where D plate spacing cm A plate contact area cm2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 3 PA 3000 User Manual Additionally conductive properties are also described as conductivity Conductivity is simply the reciprocal of resistivity Conductivity 1 and is expressed in the units siemens cm The reason for using resistivity or conductivity to describe the conducting properties of a material is that they are independent of cuvette electrode spacing and the electrode area in contact with the material The resistance however is dependent on the physical dimensions Standard cuvettes have fixed separation between plates e g 1 2 or 4 mm and fixed electrode areas of 1 or 2 cm2 The same sized cuvette filled to different volumes would result in samples with different resistances due to the different area of electrode contact The table below shows resistivity and resistance data for standard cuvettes filled with phosphate buffered saline PBS and distilled water DW Incidentally DW is one of the most resistive least conductive solutions Table of Resistance Load 2 cm Resistance Cuvette and Volume 1 mm with 50 l 2 mm wit
18. 5 0990 1 1 PA 3000 Users Manual Back Panel Symbols Protective Caution refer Caution risk of Chassis Conductor to documentation electric shock Ground Terminal The PA 3000 is rated for operation with line mains voltage 100 240 VAC maximum current 2 amps frequency 50 60 Hz There are additional fuses internal to this unit operator non replaceable rated 2 amps 250 volts The AC mains power supply cord is the disconnect device for this product The power supply cord shall be an approved cord set Type SJT rated 300 Volts AC 18 AWG 105 C 3 conductor including ground This unit is rated for environmental conditions of 5 40 C maximum relative humidity 80 for temperatures up to 31 C decreasing linearly to 50 relative humidity at 40 C altitude to 2000 meters There are no operator replaceable parts inside the system Cyto Pulse recommends that the user not remove the cabinet covers Cyto pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 1 2 PA 3000 User Manual 2 Tutorials This chapter presents tutorials on electroporation and the various types of equipment used in electroporation 2 1 Electroporation Electroporation is the name for the use of a trans membrane electric field pulse to induce an effective state of poration in a bio membrane The pores formed by this process are commonly called electropores Their presence allows molecules ions and water to
19. 51 982 8 Hashimoto K Tatsumi N Okuda K 1989 Introduction of phalloidin labeled with fluorescein isothyocyanate into living p olymorphonuclear leukocytes by electroporation J Biochem Biophys Methods 19 143 154 9 Inoue K Yamishita S Hata J Kabeno S Asada S Nagahisa E Fujita T 1990 Electroporation as a new technique for producing transgenic fish Cell Differ Dev 29 123 128 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 6 5 PA 3000 Users Manual Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 6 6 PA 3000 Users Manual Chapter 7 Service 7 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 THI
20. CA Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 B 6 PA 3000 Users Manual Appendix C Declarations of Conformity Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 1 PA 3000 Users Manual Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 PA 3000 Users Manual Replace this page with CE Certificate Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 3 PA 3000 Users Manual Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 4 PA 3000 Users Manual Replace this page with FCC Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 5 PA 3000 Users Manual Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 6 PA 3000 Users Manual Appendix D System Operation Flowchart Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 D 1 PA 3000 Users Manual Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 D 2 Power On System Time Set Protocol Select Edit Run Protocol Replay Edit Edit Replay Top Level start 1 start 2 Protocol Select Edit Group Edit Protocol lt N gt Protocol Menu Protocol
21. Cyto Pulse Sciences I nc TM Model PA 3000 Rectangular Wave Electroporation System User Manual October 2004 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 410 715 2148 FAX e mail customer 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 7 1 The LIMITED WARRANTY SET FORTH HEREIN 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 The equipment is not approved by the FDA for use to perform in vitro or in vivo diagnostics or therapy The information in this manual is subject to change without notice Copyright 2004 Cyto Pulse Sciences Inc Price 100 00 ii PA 3000 User Manual Table of Contents page 1 Introduction 1 1 2 Tutorials 2 1 2 1 Electroporation 2 1 2 2 Equipment 2 2 2 2 1 Electric Fields in Aqueous Solutions Load 2 2 2 2 2 Exponential Decay 2 4 2 2 3 Square Wave Rectangular Wave 2 5 2 2 4 Cyto Pulse PA 3000 Pulser 2 5 2 3 Protocols 2 6 3 PA 3000 Overview 3 1 3 1 Overview 3 1 3 2 Very Important concepts 3 1 3 2 1 Load 3 2 3 2 2 Relationship Between Power Supply and Pulse Voltage 3 2 3 2 3 Pre Pulse Load Estimator 3 3 3 2 4 Pulse Dr
22. ED Ready LED will be illuminated NEVER OPERATE THE SYSTEM WITHOUT A CUVETTE FILLED WITH AQUEOUS SOLUTION INSTALLED IN THE CUVETTE HOLDER 4 2 Pulse Generator There are no connections to be made to the pulse generator front panel The three functions on the front panel are the Line Power on off switch the system Reset red push button and the indicator LEDs light emitting diodes The LEDs indicate the equipment status A description of the front panel functions is given in Chapter 5 There are several back panel connections required They are Line Power Cord The line cord supplied with a three prong IEC connector must be plugged into the back of the unit DO NOT CONNECT THE OTHER END OF THE LINE CORD TO THE WALL UNTIL ALL INSTALLATION IS COMPLETE High Voltage Cable the high voltage coaxial cable from the cuvette holder must be plugged into the Pulse Out MHV connector There is only one such connector on the back panel CAUTION INSURE THE HV OUT MHV CONNECTOR IS USED Cuvette Interlock Cable The interlock cable must be connected to the phono jack at the top of the back panel see section 3 3 2 above Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 4 1 PA 3000 User Manual Ground Stud It is good practice to ground electronic equipment A wire from the ground stud on the back panel connected to any good earth ground such as a water pipe is satisfactory P VMON Pulse Voltage Monitor
23. Front Panel Functions 5 1 5 2 Example Three Group Protocol 5 2 5 3 Scope V Mon and I Mon 5 9 5 4 Data Log Example 5 9 v 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 Opening the enclosure 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 button on the front panel If there is any question about the operation of this instrument call Cyto Pulse Customer Service 410 715 0990 customer cytopulse com vi PA 3000 Users Manual Chapter 1 Introduction Electroporation has many uses in the fields of cell biology medicine and microbiology and new uses are being discovered at a rapid pace In addition to the many in vitro uses for electroporation new in vivo uses such as gene therapy and chemotherapy using electroporation also are being developed Until now the capabilities of commercial electroporators have not kept up with the needs and demands of modern electroporation The PA 3000 is a flexible laboratory electroporator that provides all of the standard rectangular pulse protocols available from
24. G 2 indicates that we are programming the second group Push Down until 1 appears Then use the Cursor button to move to the next position and Push Up until 2 appears The new panel should read V 120 v Note that you can only program a voltage for Group 2 that is lower that Group 1 Press the Enter button to move to the next screen Step 10 Set pulse width Use the cursor key and the up and down arrow keys to set the panel as shown below Press the Enter button to move to the next screen G 2 gt gt PULSE WIDTH lt lt W 0 010 ms HI Cyto pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 5 PA 3000 Users Manual Ch 5 Step 11 Set the pulse interval Use the Up button to set a value of 0 50 second as shown below Press the enter button to move to the next screen G 1 gt gt PULSE INTERVAL lt lt I 0 500 sec HI Step 12 Set the number of pulses Use the cursor key and the Up and Down arrow keys to set the panel as shown below Press the Enter button to move to the next screen G 1 gt gt OF PULSES lt lt N 6 pulses HI Step 13 End the programming gt gt Protocol X Menu lt lt Next Run Protocol Press Enter to begin the HV charging procedure Step 14 Charge system At this point the PA 3000 charges the storage capacitor to the voltage setting for the Group 1 A non adjustable delay of six seconds is built in to allow voltage stabilization 4
25. S 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 7 2 Customer Service If the user believes that there is a defect in the CYTO PULSE product the customer should call CYTO PULSE Customer Service on 410 715 0990 elsewhere or contact CYTO PULSE s local 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
26. Select Return to Main Delete Protocol Add Edit Group New Protocol Load Protocol Save Protocol Run Protocol Start 1 End Start 2 Protocol Menu 9 8 7 4 5 6 3 2 1 Menu Item Return to protocol select Always Return to Main Delete protocol upon confirmation and return to protocol select Saved protocol Delete Protocol Delete last group of protocol Group count gt 1 Delete Last Group Begin group edit N 1 to group count Edit Group lt N gt Increase group count and begin group edit procedure Group count lt 3 Add New Group Ask user for save slot and further confirmation for slots already in use Always Save Protocol Begin pulsing mode Always Run Protocol Description When Visible Title Group Edit Set Voltage Pulse Width Pulse Interval Number of Pulses Start End Run Protocol HV Charge 6 sec Active Pulsing Calculated Load Push Go 90 sec Start End Time Out Go
27. after the 1 push Down until 5 appears The new panel should read W 0 050 milliseconds The HI indicator means that the system is operating in High Range If a voltage of 400V or less was input for Group 1 then the system would put itself into Low Range Press the Enter button to move to the next screen Step 6 Adjust the pulse interval G 1 gt gt PULSE INTERVAL lt lt I 0 125 sec HI Use the default value of 0 125 sec Since there is only one pulse for this group the value set indicates the interval from this pulse to the first pulse of the next group Press the Enter button to move to the next screen Step 7 Set the number of pulses G 1 gt gt OF PULSES lt lt N 1 pulses HI Use the default value of 1 pulse Press the Enter button to move to the next screen Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 4 PA 3000 Users Manual Ch 5 Step 8 Add another group gt gt Protocol X Menu lt lt Next Run Protocol The protocol selection menu appears after the first group has been completed and the X indicates that it is a new protocol not yet saved to memory To add a second group press the Up button until Add New Group appears then press the Enter button to move to the next screen gt gt Protocol X Menu lt lt Next Add New Group Step 9 Set the Charge Voltage for Group 2 G 2 gt gt SET VOLTAGE lt lt V 600 v HI The
28. age above critical voltage and 2 is applied after the first pulse Little is known about what effect second and subsequent pulses have on the cell s pore size or number Multiple pulses are reported to give better results than single pulses in many protocols For practical purposes follow up pulses should be the same width or narrower than the first pulse 2 4 5 Movement of material into cells Two forces are known to affect transport of molecules into cells One is electroosmosis This force occurs as a result of charge differences between the cell membrane within the pore and water molecules adjacent to the charged membrane The membrane is negatively charged As a result the layer of water immediately adjacent to the cell membrane is positively charged This results in movement of water within the pore toward the negative electrode Movement of water into the cell on one end and out or the cell on the other end pulls dissolved molecules in the direction of water transport The other known material transport force is electrophoresis Negatively charged molecules such as DNA move toward the positive electrode opposite to the direction of electroosmosis This force is linearly proportional to the voltage and time of voltage application This means that the best transport by electrophoresis occurs in high voltage fields that are applied continuously There are important factors such as heat production that limit the voltage and the duration of vo
29. age of the pulse that will appear across the cuvette However it is close and the actual pulse amplitude can be estimated if the value of the load resistance is known The circuit presented in Figure 3 2 explains the problem As shown there are really two types of resistances those inside the box and one outside the box i e aqueous solution in cuvette The resistances inside the box called source resistance Rs is the inherent resistance in the high voltage switch and an additional resistance included to preclude excessive current from flowing if the output is inadvertently shorted The total source resistance is usually a few ohms Again from Ohms Law I total Power Supply Voltage Rsource Rload From this relationship the voltage which appears across the load is always less than the power supply voltage The Pulse Amplitude is given by High Voltage Switch Power Supply Rsource Internal Rload Aqueous Solution in Cuvette HV Capacitor SetV Pulse Amplitude at cuvette Figure 3 2 External and Internal Resistance Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 3 2 PA 3000 User Manual Pulse Amplitude volts I total amps x R load ohms Power Supply Voltage x R load R load R source Thus the voltage is divided between the source resistance and load resistance As the load resistance goes to zero so does the pulse amplitude voltage Figure 3 3 below illustrates the typic
30. al are the same as the high voltage current pulse The pulse rise time out of this monitor is slower than the actual pulse current rise time If pulse current rise time measurements are critical than an external torroidal type current transformer should be used see below external measurements In addition to pulse voltage and pulse current two other parameters of interest may be calculated resistance of the external load buffer in cuvette or tissue and charge The resistance in vitro or in vivo is calculated by External Resistance Pulse Voltage Pulse Current ohms The total charge transferred by a squarewave pulse is calculated by Total Charge i t dt 0 PW I PW in coulombs where I flattop pulse current in Amps PW pulse width in seconds Combining with the current monitor equation above Total Charge Current Monitor x 20 x Pulse Width coulombs Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 B 2 PA 3000 Users Manual B 2 Internal Pulse Voltage and Current Monitors The Pulse Voltage Monitor and Pulse Current Monitor signals are derived from the high voltage pulse The circuit diagram is shown in Figure B 1 below Equivalent Series Resistance in Reservoir Capacitor Current Viewing Resistor CVR 0 20 ohms System Ground Earth 10 k h 50 ohms Protection Resistors 2 2 ohms High Voltage Switch from High V
31. al power supply versus pulse amplitude relationship 3 2 3 Pre Pulse Load Estimator The load is a function of the material in the cuvette the cuvette spacing and the cuvette plate contact area The user may not know these parameters In order to estimate the load so an estimate of pulse amplitude can be provided the PA 3000 uses a pre pulse that is generated by the computer before the high voltage is turned on and the protocol is started This pre pulse is 2 2 s in duration and 2 4 volts in amplitude The pulse is placed across the cuvette plates and the resulting current measured Since the voltage is known the resistance may be calculated by the microprocessor The result is presented in the DATA LOG as resistance in ohms and conductance in siemens see Figure 5 4 Pulse Voltage Power Supply Voltage vs Load Load Resistance ohms 10 100 1000 Pulse Ampliutde Power Supply Voltage 0 80 0 82 0 84 0 86 0 88 0 90 0 92 0 94 0 96 0 98 1 00 Figure 3 3 Pulse Amplitude versus Load Resistance Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 3 3 PA 3000 User Manual The Pre pulse also is used to detect possible fault conditions such as excessive ionic solutions If the load estimate is less than 8 ohms the high voltage will not be enabled and an External Fault will appear 3 2 4 Pulse Droop As explained in section 2 2 3 rectangular wave porators also use storage or
32. aly induced DNA uptake by cells is a fast process involving DNA electrophoresis 1991 Biophys J 60 804 811 3 Antonov PA Maximora VA Pancheva RP Heat shock and osmotically independent steps by DNA uptake after Escherichia coli electroporation Biochim Biophys Acta 1993 1216 2 286 288 4 Sowers AE Mechanisms of electroporation and electrofusion in Guide to Electroporation and Electrofusion Editors Chang Chassy Saunders and Sowers 1992 Academic Press 119 138 5 Nickoloff Jac A ed 1995 Electroporation Protocols for Microorganisms Methods in Molecular Biology Volume 47 Humana Press Totowa New Jersey 372 pp 6 Nickoloff Jac A ed 1995 Animal Cell Electroporation and Electrofusion Protocols Methods in Molecular Biology Volume 48 Humana Press Totowa New Jersey 369 pp 7 Sowers A E 1995 Permeabiliy alteration by transmembrane electric fields electroporation IN Permeability and Stability of Lipid Bilayers E A Disalvo and S A Simon eds CRC Press Boca Raton p 105 121 8 Chang D C Chassy B M Saunders J A and Sowers A E eds 1992 Guide to Electroporation and Electrofusion Academic press San Diego 581 pp 9 Dimitrov D S and Sowers A E 1990 Membrane electroporation fast molecular exchange by electroosmosis Biochimica et Biophysica Acta 1022 381 392 10 Neuman E Sowers A E and Jordan C A eds 1989 Electroporation and Electrofusion in Cell Biology Plenum Press New York
33. and given the symbol F which is number of electrons per volt The pulse width is dependent on the size of the capacitor and the resistance load of the medium solution or tissue The pulse shape is a double exponential with a very fast rise time and a slow exponential decay fall time The width at the 50 of amplitude point is given by Width 50 0 7 x C farads x R ohms For example if an ED electroporator has a 500 F reservoir capacitor and discharges into a 2 mm cuvette filled with 200 l PBS resistance of 12 ohms the pulse width at the 50 of amplitude point is about 6 milliseconds Below is a graph showing waveforms for a 50 F 500 F and 5000 F reservoir capacitor and a 16 ohm Load Resistance The waveform follows a standard exponential or half time decay Time From Pulse Start milliseconds 0 50 100 150 200 Voltage volts 0 200 400 600 800 1000 5000 F 50 F 500 F Figure 2 6 Pulse Amplitude vs Time The Exponential Decay pulser although inexpensive is a crude device As can be seen from the above example the amplitude needed for electroporating is in the early portion of the pulse but the total area under the curve contributes to heating the sample Also the pulse width is dependent on the conductivity of the solution or tissue being porated without compensation changes from one experiment to the next will cause the pulse width to change In addition si
34. arted This chapter includes some examples to get started 6 1 Checklist The Following checklist will help you begin your electroporation optimization Check List for Electroporation Optimization beginning point 1 What is the cell type used 2 What is its diameter 3 Published Pulse voltage 4 Published Pulse width 5 Published number of pulses 6 Calculated threshold voltage 7 What is the conductivity of the medium 8 Desired cell viability 9 Desired electroporation efficiency 10 Molecule for electroporation DNA dye etc 11 Cuvette electrode gap 12 V cm X electrode gap 13 Percent of voltage delivered to cuvette 14 Set voltages Explanation of questions 1 Self explanatory 2 Can be estimated Red blood cells are approximately 7 microns lymphocytes are approximately 10 microns K562 cells are approximately 20 microns See chart below for more examples 3 If known See 6 1 Published Protocols for some examples 4 If known See 6 1 Published Protocols for some examples 5 If known See 6 1 Published Protocols for some examples 6 Use the formula Vm 1 5 rE cos B where r is the radius of the cell E is the strength of the external field B is the angle between the direction of the external field and the normal vector of the membrane at the specific site and Vm 1 for the breakdown threshold voltage Solving for E and using a cosine of 1 then E 1 1 5 radius in mic
35. d 50 micro seconds Note that for any given cell size there is a wide range of voltages used To see if the value that we have chosen is within a reasonable value for the cell size we will calculate the external voltage from the formula Vm 1 5 rE cos B where r is the radius of the cell E is the strength of the external field B is the angle between the direction of the external field and the normal vector of the membrane at the specific site and Vm 1 for the breakdown threshold voltage Actual threshold for membrane breakdown ranges from 0 2 volts to 2 volts across the cell membrane One volt is commonly used as the average threshold voltage thus the selection of Vm 1 Vm 1 1 5 15 E 1 at the poles of the cell Solving this equation for E yields E 1 1 5 15 1 E 1 V 22 5 0 0444 V or 444 V cm Note that this voltage is less than the published voltage However this is the minimal threshold voltage for the average diameter cell at the poles nearest the electrodes Values several times the minimum threshold are often required If multiple pulses are anticipated pulse width should be no wider than that indicated in figure 3 4 For PBS or cell culture medium using a 100 l volume in a 2 mm cuvette the limit is approximately 200 s This limit is needed to prevent a continual drop of voltage due to failure to fully re charge the capacitor between pulses For most protocols pulse widths between 10 and 100 s and numbe
36. erent in DNA expression 2 5 4 Cell Viability In addition to choosing a method for measuring yield a method for measuring cell viability is needed Methods include 1 colony formation colony count before and after electroporation 2 trypan blue dye uptake hours after the electroporation 3 simple cell counts on tissue culture plates the day after electroporation 4 vital dye uptake of cells attached to a plate 24 hours after electroporation followed by an absorption reading of eluted dye 5 Alimar blue or other metabolic dyes and 6 flow cytometric analysis or other fluorometric analyses of Calcein AM dye uptake and 7 tritiated thymidine uptake There are many more methods and any will do although the gold standard is colony formation Keep in mind that vital dyes will penetrate permeabilized cells for some time after electroporation and cells that take up the dye may not be dead 2 5 5 Electrical Parameters There are at least two methods for choosing initial pulse parameters for electroporation protocols They are 1 Adapting to existing protocols and optimizing from this starting point 2 Using cell diameter as a starting point 2 5 5 1 Published Protocols If you have a protocol that you have developed or a protocol that others have published start with those protocol values It is more complicated to adapt an exponential wave protocol to PulseAgile in comparison to rectangular wave protocols The adaptation of expone
37. ficient Divide the range into equal parts of 25 50 volts and test the effect of each voltage on viability Pulse widths in the range of 10 to 100 microseconds should be a good starting point It may be important to start with higher initial cell viability than needed to compensate for changes made to the protocol during optimization Further optimization by changing the pulse width and number of pulses can be done at this time but it is a good idea to wait until follow up lower voltage pulses have been optimized As soon as more than one pulse is added to the protocol either as initial pulses follow up pulses or material transport pulses a pulse interval needs to be chosen A good initial interval is 125 milliseconds Note that in rectangular wave or in PulseAgile protocols pulse intervals are usually in milliseconds and pulse widths are usually in microseconds 2 5 7 Optimize multiple high voltage pulses More than one high voltage pulse may be needed Often 2 to 6 pulses are optimum These pulses can be of the same voltage as the first pulse or lower than the first pulse but still above threshold voltage It is most efficient to optimize follow up pulses using a factorial analysis design varying pulse voltage and pulse number simultaneously 2 5 8 Optimize material transport Material transport pulses are designed to move charged molecules into cells after pores have been induced The electric field of the material transport pulses
38. gile calcein molecules cell x 10 6 Figure 2 7 Example Uptake Calcein Fig 2 7 Molecular uptake of calcein a fluorescent tracer molecule is enhanced with PulseAgile protocols compared to a single pulse protocol The single pulse protocol was applied at 3 3 kV cm for 50 s The PulseAgile protocols included a single pulse 3 3 kV cm 50 s followed 0 125 seconds later by 10 pulses of 1 ms and 0 4 kV cm with interval of either 0 125 s or 20 sec DU 145 prostate cancer cells were used at 2 X 106 cells ml in a 2 mm gap cuvette and 10 M calcein Molecular transport and cell viability were calculated using calibrated flow cytometry with propidium iodide as the viability stain Mark Prausnitz Ph D Georgia Institute of Technology provided data for this and the following graph 0 50 100 Single Pulse PulseAgile cell viability Figure 2 8 Viability Fig 2 8 Cell viability was shown not to decrease with the PulseAgile protocols The pulse protocols used were the same as those used in Fig 2 7 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 13 PA 3000 User Manual 2 6 References 1 Sukharev SI Klenchin VA Serov SM Chernomordik LV and Chizmadzhev YA Electroporation and electrophoretic DNA transfer into cells The effect of DNA interaction with electropores 1992 Biophys J 63 1320 1327 2 Klenchin VA Sukharev SM Chernomordik LV Chizmadzhev YA Electric
39. h using PulseAgile protocols 2 4 8 Other Cell Associated Factors Other cell specific factors add to variability in electroporation efficiency Cell cytoskeletal structure is an example Increased density of cell cytoskeleton at the site of pore formation can make the cell more resistant to detrimental effects of excessive pore expansion Roughness of the cell due to cell processes or villi are another example 2 4 9 Solution Temperature Pore Closing Times The temperature of the cell membrane or medium influences pore life span Cell membrane pores remain open for seconds to minutes at room temperature Higher temperatures accelerate pore closure Alternatively at 4 oC cell membranes are viscous and inflexible and pore closure is slower Pore induction or formation is similarly affected by temperature variations It is more difficult to induce pores in cold cell membranes For maximum pore life cells would be electroporated at 27 37 oC and brought rapidly to 4 oC These methods of prolonging pore life are rarely practical 2 4 10 Miscellaneous Addition of Reagents Electroporation efficiency is much higher if the molecules that you want to introduce into cells DNA proteins small molecules are in the cell solution before application of pulses rather than after Even though electropores are theoretically open for seconds to minutes close association of DNA with cells at the time of electroporation is essential 2 5 Method Developme
40. h 200 l 4 mm with 800 l PBS1 60 25 oC 12 12 12 Distilled Water2 18 Meg 3 6 Meg 3 6 Meg 3 6 Meg 1 Sigma PBS cat D8662 2 Sigma water cat W3500 2 Resistivity is a strong function of temperature value given at 25 oC If a 1000 volt pulse is applied to a cuvette with a 2 mm spacing and 200 l PBS buffer the current that will flow is 1000 volts 12 ohms 83 amps 2 2 2 Exponential Decay ED Electroporators The simplest approach to generating a high voltage pulse is to charge a capacitor C with a high voltage power supply and then discharge the capacitor into the chamber containing the cells in the desired aqueous medium or buffer The cells and the buffer represent the electrical load or resistance R for the high voltage pulse see Figure 2 5 The charge switch is shown closed and the discharge switch is shown open When the sample is to be pulsed these switch positions are reversed and the discharge switch remains closed until the capacitor is completely discharged This capacitor is also called a Resistance of Material in Solution Load Reservoir Capacitor High Voltage Power Supply Charge Discharge Switch Figure 2 5 Exponential Decay Generator Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 2 4 PA 3000 User Manual reservoir capacitor The number of electrons that the capacitor can store size is measured in farads
41. he heat produced under the same condition is halved In practice multiple wide low voltage pulses are used to induce Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 2 8 PA 3000 User Manual transport of material by electrophoresis after pores are formed by shorter high voltage pulses See section 3 1 5 to calculate temperature increase in an electroporation cuvette 2 4 7 Cell Viability Factors Excess Voltage Pulse voltages much beyond breakdown threshold result in formation of pores too large to spontaneously repair As a result cells lyse or die from loss of cytoplasmic content In a cell suspension composed of uniform diameter cells reducing the voltage readily solves the problem of extreme cell death due to excess voltage In most cell suspensions the diameter of individual cells does vary and there is a distribution of cell sizes Because of this some cell death is inevitable The larger cells will be killed as the optimal voltage for average cells is applied Conventionally maximum poration has been observed using pulse parameters where about half of the cells are killed This is because traditional protocols use the same pulse conditions for material transport as those to initially form the pores PulseAgile capability allows separation of desired effects with resultant increases in efficiency and less cell death For example in K562 cells we have achieved 40 transfection with less than 10 cell deat
42. is lower than the first pulses Values at or below threshold are used All further optimization should focus on yield and cell viability simultaneously It is important to monitor both yield and cell viability to be able to identify positive or negative trends in electroporation efficiency Choose a range of voltages to be tested Values of one half one fourth one eighth and one sixteenth of the voltage of the first pulse are reasonable starting values Choose a range of pulse width s to be tested for each voltage Start with a range of 200 microseconds to 2 milliseconds Use multiple pulses to start with to save time in the optimization since multiple pulses will often be used in the final protocol Four pulses is a good Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 2 12 PA 3000 User Manual starting point With a fixed number of pulses and pulse width test the effect of changing voltage through the range of voltages Next test the range of pulse widths against the optimal voltage Repeat this process until an optimum is found Again employing a factorial analysis by modifying electric field pulse width and pulse number simultaneously may save time The reason for the increased efficiency is that with a factorial design interactions can be assessed and experimental variability is measured over the entire assay rather than just repetitions at individual independent variables 0 1 2 Single Pulse PulseA
43. item is the current date and time Protocol Select Open an existing protocol from memory or begin a new protocol from scratch Protocol Menu Allowable operations on an existing protocol run the protocol save delete the protocol add remove edit groups and return to protocol selection Set pulse width Pulse width is displayed as milliseconds so a pulse width of 50 microseconds is displayed as 0 050 The range of values possible is 1 microsecond 0 001 to 2 milliseconds 2 000 incremented in one microsecond steps Set pulse interval Pulse interval is the time from the beginning of one pulse to the beginning of the next pulse The pulse interval is displayed in seconds The range of values is 0 125 seconds to 400 seconds in 0 001 second steps Set number of pulses The number of pulses ranging from 1 to 99 per group Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 2 PA 3000 Users Manual Ch 5 5 4 An Example protocol The following is an example protocol consisting of two Groups Group No Voltage Width Interval Number 1 600 0 050 0 125 1 2 120 0 010 0 50 6 Step 1 Turn on the PA 3000 The on off switch is in the lower left corner of the front panel Step 2 Adjust the time and date Adjust System Time 10 05 02 7 08 01 25 Push the cursor button to toggle among the choices The date is represented in the form MM DD YY DoW If the date appears as show
44. l called External Fault will be illuminated If this fault occurs the cuvette or chamber must be examined to see what caused the fault Typically using high pulse repetition rates wide pulse widths and small aqueous solution volumes will cause this type of condition Internal Fault Detection A number of internal circuit checks are made before the high voltage is turned on and during operation If any of these checks detect an anomalous condition the system will shut down and the Internal Fault LED will be illuminated There are two types of internal faults Average Current Exceeded The high voltage power supply has a fixed average current limit If this limit is exceed an internal fault will occur This can occur by turning the power supply voltage knob too fast spinning or by attempting to run pulse protocols which are outside of the limits of the PA 3000 high repetition rates large number of pulses with wide pulse widths Equipment Component Failure This type of fault is rare First determine if the internal fault occurred because of excessive average current by running a protocol with a single 50 s pulse If the internal fault still occurs call technical support and have available the exact conditions including protocols which were being used 3 4 System Specifications The full specification set is presented in Appendix A Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 3 5 PA 3000 User Ma
45. l purposes after about 3 seconds the pores are closed to movement of large molecules into cells 2 4 Optimizing PulseAgile Protocols The optimization process should proceed iteratively modifying one variable at a time The following is a general outline for optimizing protocols 2 4 1 Background There are several components to PulseAgile protocols It helps to breakdown the optimization process into parts in order to address the variables and avoid becoming overwhelmed by the number of possible combinations An electroporation protocol can be broken down into three parts 1 First pulse to begin initial pore formation 2 Follow up high voltage pulses to yield further pore formation 3 Material moves into the cell Other factors that influence the electroporation process are 1 Cell viability factors 2 Brownian movement and vector considerations 2 4 2 Initial Pore Formation When an external electrical potential is applied to a cell the cell membrane resists breakdown until a critical threshold voltage is achieved As the voltage reaches the threshold the cell membrane ceases to resist and a pore is formed in the cell membrane The breakdown voltage is roughly one volt across the cell membrane Mathematically voltage at the cell membrane is defined as Vm 1 5 rE cos B where r is the radius of the cell E is the strength of the external field B is the angle between the direction of the external field and the normal vect
46. l this be a one time use What other factors are important Using this list you should be able to choose the desired result For instance if the desired goal is generating a clone of cells from a group of cells transfected with the same plasmid the percent of viable cells need not be high If the goal is genetic engineering of rare primary cells cell viability is very important From this evaluation you should be able to answer important questions regarding your electroporation goals 2 5 2 Electroporation Medium As with many factors in electroporation choice of medium involves compromise Voltage drop during the pulse and heat generation is easily controlled when using high resistance low ionic medium High resistance medium is usually composed of water with a non ionic buffer and a sugar or sugar alcohol added to adjust osmolarity to as close to 290 milliosmoles as possible However some cells do not survive well in low ionic medium For example sugars are often toxic to mammalian cells during electroporation Many different types of molecules are exchanged between the cell cytoplasm and the medium during electroporation For this reason mammalian cells are usually placed in an ionic medium for electroporation Cell culture medium or a buffered salt solution is often used Addition of ions that are normally at a high concentration inside the cell and a low concentration outside the cell may improve cell viability Addition of potassium and
47. lse Amplitude BNC connector Scale voltage x 200 volts System Ground bolt connector to a local ground Detail 2 Pulse trigger BNC connector Used as an oscilloscope trigger signal when viewing the pulse monitor Cuvette Interlock The plug from the mating phono plug from the cuvette holder must be inserted for the system to operate Serial Interface DB9 connector Connect to a computer to download the log from the microprocessor Serial Number Label Location Detail 3 System Fuse Holder Main Line Power Entry Cy PA 3000 Users Manual Ch 5 Chapter 5 Instrument Operation 5 1 Introduction All of the control functions for the PA 3000 are on the front panel The figure below shows the layout of control functions displays and dials On the left of the control panel are six buttons that are used for programming the electroporation protocol Above the six buttons is the liquid crystal display that shows the input parameters On the lower left of the front panel is the line power switch In the center of the panel near the bottom is the voltage adjustment knob The right side of the panel has Light Emitting Diodes LED to indicate the status of various functions of the PA 3000 On the lower right hand side of the panel is an emergency shutoff reset switch Control Buttons Stop Reset Status LEDs ON OFF Liquid Crystal Display Fault LEDs Cyto Pulse Sciences Inc Figure 5 1 Front Pa
48. ltage application that can be applied to cell suspensions Generally the most practical and effective mass movement derived from electrophoresis is obtained when lower voltages are applied in multiple medium to long length pulses One publication suggested that all effective movement due to electrophoresis occurs within 3 seconds of the original pulse That time limit can serve as a guideline 2 4 6 Cell Viability Factors Heat One important limit to the length of time that voltage and the resultant current can be applied to cells is heat production within the solution Heat production is exponentially proportional to electrical current within the solution After pulses are applied there is some cooling within a solution due to a heat sink effect from the relatively large mass of metal in electrodes However the cooling is not rapid enough to compensate for the rapid rise in temperature related to excessive electrical current during the application of pulses One method to compensate for heat production due to electrical current is to reduce the applied voltage and deliver wider pulses While heat reduction is exponentially related to voltage reduction the loss of movement by electrophoretic force is only linearly related Movement due to electrophoresis is accomplished by electrical charge For example a reduction of the voltage by half coupled with a simultaneous doubling of pulse width results in the same movement of material by charge T
49. ltage up to the set voltage Otherwise the voltage will continually drop between pulses Failure to stay within these limits will result in the delivery of pulses with unintended voltages 3 2 6 Aqueous Solution Heating Maximum Pulse Width vs Load droop lt 5 Load ohms 10 100 1000 Maximum Pulse Width microseconds 100 1000 In general operating conditions should be maintained that minimize heating of the aqueous solution in the cuvette There are a number of variables involved which contribute to temperature raise 1 The longer the pulse 2 The narrower the space in the cuvette 3 The lower the load resistance 4 The shorter the interval between pulses The best way to monitor heating of the aqueous solution in the cuvette is using an oscilloscope Connect the scope to the pulse current monitor port on the back of the unit I MON Monitor the flattop of the current pulse If Figure 3 4 Maximum Pulse Width versus Load Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 3 4 PA 3000 User Manual the flattop increases during the pulse there is significant heating in the cuvette See Appendix C for details 3 3 Safety Features There are many safety features designed into the equipment to protect the users and the machine Four of those are visible to the user and will be described below Cuvette Holder The Cuvette Holder was designed to prevent accidental contact with
50. mplitude P VMON 200 1 200 volts volt 5 Pulse Current P IMON 20 1 20 Amps volt 5 Safety Load check before high voltage enabled Shut down if pulse current gt 125 Amps arc Shut down if internal malfunction Cuvette interlock high voltage disabled if cuvette holder open Maximum Pulse Values vs Load Power Supply Voltage 1100 volts and Pulse Number 99 Rload Pulse V Pulse I Pulse Width microseconds ohms Volts Amps Rate pulses per second 1 2 4 8 10 936 94 267 133 66 33 20 1011 51 494 247 123 61 40 1054 26 948 474 237 118 60 1069 18 1403 701 350 175 80 1076 13 1857 928 464 232 100 1081 11 2000 1156 578 289 200 1090 5 2000 2000 1146 573 400 1095 3 2000 2000 2000 1141 800 1098 1 2000 2000 2000 2000 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 A 2 PA 3000 Users Manual Appendix B Pulse Voltage and Current Measurements This appendix describes how to make pulse measurements using the internal monitors or by using external customer supplied equipment which will improve accuracy by a few percent Both of these techniques require the use of an oscilloscope that usually has a measurement accuracy of 3 to 4 in addition to the above Also described are the internal circuits of the monitors and how the scale factor is derived A Tektronix digital oscilloscope TDS 210 may be purchased from Cyto Pulse
51. n above and the desired date is November 12 2004 Friday i e 11 12 04 6 then do the following Push the cursor key to move to the second position then press the Up button to change the second digit to 1 Move to the day and press Up until 08 is displayed Move to the day of week position and set 6 by pressing the Down button Move the cursor to the time and set the current time Press the Enter button to move to the next screen The next time the system is turned on the proper date and time will be displayed Step 3 Begin a new protocol gt gt PROTOCOL SELECT lt lt Next New Protocol By default a prompt will appear to begin a new protocol If previously saved protocols exist in any of the PA 3000 s ten memory slots pressing the Up and Down buttons will scroll through them for quick retrieval For the purpose of this example however press the Enter button to begin a new protocol entry and move to the next screen Cyto pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 3 PA 3000 Users Manual Ch 5 Step 4 Set the Charge Voltage for Group 1 G 1 gt gt SET VOLTAGE lt lt V 300 v The G 1 indicates that we are programming the first group Push Up until 6 appears The new panel should read V 600 v Press the Enter button to move to the next screen Step 5 Adjust the Pulse Width G 1 gt gt PULSE WIDTH lt lt W 0 100 millisec HI Move the cursor under the 0
52. nce the capacitor is totally discharged it must be totally recharged before it can be used again This will limit protocols where multiple pulses are required Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 5 PA 3000 User Manual 2 2 3 Rectangular Wave Electroporators The next level of sophistication in generating pulses is achieved by using a high voltage solid state switch that turns on only for the desired pulse duration This system still has a reservoir capacitor but it is only discharged by a few percent This approach permits the pulse width to be set to any value desired by the user The pulse width delivered to the chamber is now independent of chamber resistance and the pulse amplitude remains relatively constant during the time the pulse is on These together provide a more repeatable electric field pulse In addition since the capacitor is only discharged a few percent the voltage on the reservoir capacitor can rapidly be brought up to full value permitting multiple pulses with relatively short pulse intervals The rectangular wave pulser eliminates many of the drawbacks of the ED pulser 2 2 4 Cyto Pulse PA 3000 Rectangular Wave Pulse Generator The PulseAgile pulser provides a high level of sophistication It provides control of all protocol parameters with the ability to set pulse width amplitude time between pulses and the electric field direction For the first time researchers have the
53. nel Functions 5 2 Getting Started Operation of the PA 3000 is performed by following these steps Insert the cuvette into the holder with aqueous solution Close the cuvette holder Program your protocol Set the voltage Check the status and fault LEDs Push the start button Cyto pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 1 PA 3000 Users Manual Ch 5 5 3 Programming the Protocol 5 3 1 Groups of Pulses The basic concept of operations is a group Up to 3 groups can be programmed per protocol Within each group pulse parameters such as pulse width and pulse interval are identical The number of pulses 1 to 99 is programmed independently for each group This option is unique in rectangular wave porators To program standard protocols only one group is used Figure 5 2 gives an example of a 3 Group protocol 0 20 sec 0 13 sec 0 13 sec 0 13 sec 0 13 sec 0 13 sec 50 s Group 1 Group 2 Group 3 20 s 20 s 10 s 10 s 20 s Figure 5 2 Example Three Group Protocol 5 3 2 Menu Operation The menu is changed using the five buttons below the liquid crystal display The cursor button is used to toggle through the displayed menu item The up and down arrow keys are used to change the values of the selected item The enter button is used to accept the selected values and to move to the next menu item Set date and time The first menu
54. nt There are many combinations of pulse parameters possible using PulseAgile electroporation Similarly there are several ways to arrive at an optimal combination of the electroporation parameters The following is one way Electroporation protocols are developed iteratively The following sequence is suggested 1 Choose a starting point goals medium and reporter molecules 2 Separate the protocol into components initial pore formation follow up pulses and mass transport 3 Optimize initial pore formation 4 Optimize follow up pulses 5 Optimize mass transport 6 Repeat steps 3 4 5 if necessary and optimize other parameters if desired Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 9 PA 3000 User Manual 2 5 1 Choosing a Starting Point First choose goals for the electroporation procedure The following questions may help What molecules are you trying to get into the cell What are the characteristics of the molecules size charge in solution etc What type of cell are you using What are the cell s characteristics What is the cell size Do the cells have cell walls Do you know of any substances in the proposed medium that are toxic to the cells Is cell viability important Is electroporation efficiency important Are single clones to be selected from the cells Are cells to be part of a library Are cells to be used in bulk without cloning Will this protocol be used repeatedly or wil
55. ntial decay protocols is as follows The first pulse is of the same voltage as the peak exponential voltage with a pulse width from 10 to 100 microseconds This pulse will be the pore forming pulse The second pulse is half the voltage and two times as long The third pulse is half again the voltage and two times as long as the second pulse A fourth pulse may be optionally be added with half again the voltage and twice again the pulse width Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 11 PA 3000 User Manual 2 5 5 2 Cell Diameter If published protocols are not available for your cell type values for a similar cell type can be used or a starting voltage can be calculated using the average cell radius in microns of the cells in suspension The formula described below can be used to calculate a starting point Often multiples of the threshold voltage are used Threshold in volts cm E 10 000 15 r where r is the cell radius 2 5 6 Optimize the first pulse There are many combinations possible using PulseAgile technology and there are several ways to arrive at the optimal combination The following is one way Start with an evaluation of the effect of first pulse voltage on cell viability Pick a range of voltages to work with around the chosen starting voltage Generally twice the threshold voltage is a reasonable starting voltage A range of the starting voltage 33 50 should be suf
56. nual Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 3 6 PA 3000 User Manual Chapter 4 Setup There are several important safety and operational concepts described in Chapter 3 For your safety and for proper operation of the PA 3000 Chapter 3 must be read first DO NOT PLUG IN THE POWER CORD UNTIL ALL OF THE SET UP PROCEDURES AS DESCRIBED BELOW HAVE BEEN COMPLETED 4 1 Cuvette Holder The Cyto Pulse cuvette holder is designed to accept industry standard cuvettes The safety features are described in section 3 3 The holder has two cable assemblies one for the pulsed high voltage connection and the other for the safety interlock For the system to operate the larger cable coaxial must be plugged into the back panel of the high voltage pulse generator at the Pulse Out MHV connector There is only one connector on the back of the pulse generator which will accept the high voltage cable connector WITHOUT forcing The safety interlock cable must be plugged into the back panel of the pulse generator at the phono jack labeled Cuvette Interlock For the system to operate the interlock must be satisfied That is the cuvette handle must be all the way forward If the interlock is not closed the Cuvette Open fault red LED will be illuminated and the green Ready LED will not be illuminated If the interlock is closed then the red Cuvette Open fault LED will not be illuminated and the green L
57. of 1 0 volt is normally used Serial RS 232 Interface Optional This DB9 nine pin connector interface may be used to read the log produced by the microprocessor The log provides the time the protocol was run and the parameters used along with the estimate of load resistance Any terminal program may be used such as Hyper Terminal in Windows 95 Phone number is set to Direct to Com 1 Baud rate is set to 9600 THIS COMPLETES THE INSTALLATION PROCEDURE ANY QUESTIONS SHOULD BE DIRECTED TO TECHNICAL SUPPORT DO NOT CONNECT THE LINE CORD UNTIL THE OPERATION CHAPTER CH 5 IS READ Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 4 2 PA 3000 User Manual PA 3000 Figure 4 1 Back Panel Layout DETAIL 1 DETAIL 2 DETAIL 3 Detail 1 High Voltage Pulse output Pulse voltage Monitor Pulse Current Monitor Detail 2 Serial interface Cuvette Holder Interlock Pulse trigger out Detail 3 Line Mains input Fuse Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 4 3 PA 3000 User Manual Detail 1 Pulsed High Voltage Out Two banana jacks red HV to Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 4 4 or One MHV connector NEVER use both simultaneously NEVER force a BNC connector on the MHV Pulse Monitors oscilloscope Pulse Current BNC connector Scale voltage x 20 amps Pu
58. oltage Power Supply and Capacitor Amplifier divide by 4 Amplifier unity gain External Load Buffer Solution Tissue Pulse Current Monitor IMON Pulse Voltage Monitor VMON Back Panel Power Supply Voltage Monitor to computer and data log Voltage Monitor Scale Factor 50 10 000 1 200 Current Monitor Scale Factor 0 20 4 1 20 otal Internal Resistance 1 7 ohms T Current Monitor Scale Factor Figure B 1 Simplified Circuit Diagram of Monitors Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 B 3 PA 3000 Users Manual When the high voltage switch is closed the primary pulse current path is through the switch protection resistor the external load and the current viewing resistor From Ohms Law a voltage is present at the Current Viewing Resistor CVR Voltage Total Current x 0 2 ohms If the total current is 100 Amps then the voltage is 20 volts This is reduced to 5 0 volts by the divide by 4 amplifier Voltage Monitor Error Due to CVR The voltage monitor circuit is a resistive divider It is across the External Load plus the Current Viewing Resistor This is important because the voltage applied to the External Load is slightly less than that measured by the Pulse Voltage Monitor This error is 1 78 at the lowest value of permitted External Resistance of 8 ohms As shown in Figure B 2 as the External Load Resistance gets larger the error gets smalle
59. oop 3 4 3 2 5 Maximum Pulse Width 3 4 3 2 6 Aqueous Solution Heating 3 4 3 3 Safety Features 3 5 3 4 System Specifications 3 5 4 Setup 4 1 4 1 Cuvette Holder 4 1 4 2 Pulse Generator 4 1 iii 5 Instrument Operation 5 1 5 1 Introduction 5 1 5 2 Getting Started 5 1 5 3 Program the Protocol 5 2 5 3 1 Groups of Pulses 5 2 5 3 2 Menu Operation 5 2 5 4 An Example Protocol 5 3 5 5 System Check 5 9 6 Getting Started 6 1 6 1 Checklist 6 1 6 2 PA 3000 Protocol Optimization 6 2 6 2 1 Choosing Starting Voltage and Pulse Width 6 3 6 2 2 Cuvette Considerations 6 3 6 3 Optimization Methods 6 4 6 3 1 Quick optimization 6 4 6 3 2 Small factorial analysis 6 4 6 3 3 More detailed factorial analysis 6 5 6 4 References 6 6 7 Customer Service 7 1 Appendix A Specifications Appendix B Pulse Voltage and Current Measurements Appendix C Declarations of Conformity Appendix D System Operation Flowchart iv PA 3000 User Manual List of Figures page 2 1 Electroporation 2 1 2 2 E Field Intensity vs Pulse Voltage and Cuvette Spacing 2 3 2 3 Cuvette 2 3 2 4 Exponential Decay 2 4 2 5 Exponential Decay Pulses 2 5 3 1 Accessories 3 1 3 2 External and Internal Resistance 3 2 3 3 Pulse Amplitude versus Loan Resistance 3 3 3 4 Maximum Pulse Width versus Load 3 4 4 1 Back Panel Layout 4 3 5 1
60. or of the membrane at the specific site Since the breakdown voltage is approximately 1 volt actually 0 2 to 2 volts the critical voltage for a cell in volts micron is E 1 1 5 r at the poles where cos B 1 Multiplying this result by 10 000 gives the result in Volts cm For example for a 40 micron diameter cell the voltage needed to achieve critical voltage is 1 1 5X20 0 033 Volts per micron or 333 volts per cm In practice somewhat higher voltages are used since the calculated voltage is the minimum breakdown voltage 2 4 3 Initial Pulse width The initial pulse width needs to be long enough to allow for pore formation and short enough to prevent excessive pore expansion or heat formation A short period of time is needed for membranes to respond to the applied force Minimum times are under one Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 2 7 PA 3000 User Manual microsecond so this is not a practical limiting factor Maximum pulse width is not a precise point and depends upon the cell viability desired Over a limited range increasing pulse width is equivalent to increasing pulse voltage That is effective electroporation is proportional to the area defined by voltage X pulse width We suggest starting pulse widths in the range of 10 to 100 microseconds 2 4 4 Follow up High voltage Pulses Further pore formation A follow up pulse is defined for this manual as any pulse that 1 has a volt
61. r External Resistance ohms 10 100 Percent Error 0 1 2 3 Figure B 2 Error in Voltage Monitor Due to Current Viewing Resistor Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 B 4 PA 3000 Users Manual Voltage Across External Load If the Pulse Voltage Monitor Oscilloscope combination is not used the voltage across the External Load Resistance can be estimated The voltage across the load is Pulse Voltage across External Load R R R x Power Supply Voltage ext ext int This is a standard voltage divider relationship An estimate of R ext is given by the PA 4000 in the Ohm Meter window at the bottom of the Visual Basic software interface or at the end of the run in the PA 3000 LCD window and at the bottom of the log report The number presented is the ratio of pulse voltage to power supply voltage As the External Load gets smaller more voltage appears across the internal resistance s and less voltage appears across the External Load This is shown in the graph in Figure B 3 This ratio estimate is accurate to about 10 The R ext estimate should not be used for precise analysis Pulse Voltage vs Power Supply Voltage as a function of Load Resistance External Load R ohms 10 100 1000 Percent 70 80 90 100 Power Supply Voltage Pulse Voltage 2 IGBT 80218 b 0 40 1258061825 b 1 64 7584672745 b 2 23 8737230516 b 3 2 9449136102 r
62. r of pulses from 2 to 6 are optimum 6 3 Optimizing the Protocol The percent transfection or other electroporation result achieved by using standard published protocols may be sufficient for your needs If optimization of the protocol is needed to achieve better results there are several approaches that may be tired First a simple matrix may be tried A common number of pulses used in standard protocols is two or six pulses Use an electric field strength recommended in the literature If nothing is recommended use a starting electric field of 1500 to 2500 V cm for a 10 15 diameter cell Of course this range will change if the cell size is much smaller or bigger A starting pulse width of 50 to 100 s can be used For the matrix design a simple factorial design is recommended The following is a factorial design based upon the selected values Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 6 3 PA 3000 Users Manual Protocol Electric Field V cm Pulse Width s Number of Pulses 1 1500 50 2 2 2500 50 2 3 1500 100 2 4 2500 100 2 5 1500 50 6 6 2500 50 6 7 1500 100 6 8 2500 100 6 You also will need to do duplicate electroporations and negative controls A second or third iteration of the experiment may be needed Choose values for subsequent iterations based upon results of the immediately preceding iteration 6 3 1 Additional Cuvette Considera
63. rons X 1 To get V cm multiply the answer by 10 000 See the example shown later in this appendix Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 6 1 PA 3000 Users Manual 7 This value is important for highly conductive medium such as PBS or tissue culture medium IF highly conductive but the conductivity is unknown use the conductivity of PBS whose 60 cm 8 This value will help decide the end point for optimization 9 Again this value will help decide the end point for optimization 10 Optimization for DNA transfection may be different than that for small soluble molecules See Chapter 2 for a further explanation 11 The cuvettes come in 1 mm 2mm and 4 mm gaps The volts per cm will need to be multiplied by the inverse of the gap in cm 0 1 0 2 or 0 4 to determine the applied voltage 12 See item 11 This formula can be used for the minimum threshold voltage and all other voltage ranges used such as published voltage and maximum voltage tested 13 Use the chart on page 3 4 to determine the percent of applied voltage actually delivered at the cuvette 14 Divide all voltages listed in item 12 by the percent of voltage delivered to the cuvette item 13 Table 6 1 Published protocols Cell Line Cell diameter Voltage Pulse Width Reference CHO 20 microns 1 5 kV cm 50 s Zerbib 1985 CHO 20 microns 600 1500 V cm 100 4000 s Wolf 1994 Human RBC 7 micron
64. rtened to reduce the total energy input into the solution There are two main electroporation waveforms exponentially decaying and rectangular wave Different types of electronic equipment generate these waveforms 2 2 Electroporation Equipment 2 2 1 Electric Fields in Aqueous Solutions and Load The basic process of electroporation and electrofusion requires that cells be exposed to electric fields with special characteristics In the most elementary form the electric field can be viewed as a voltage applied to two rectangular plates with spacing between the plates see D in Figure 2 3 below The electric field is not dependent on the material between the plates As an example to a first approximation the applied electric field needed to impress a threshold voltage of one volt across a cell must be Paramecium 180 m 55 v cm Mammalian Cell 50 m 200v cm Red Blood Cell 7 m 1430 v cm Bacterial Cell 1 m 10 000 v cm More precise estimates of electric field requirements will involve the use of the so called Schwann equation For more information refer to Kinosita etal 1992 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 2 2 PA 3000 User Manual In electroporation applications a typical chamber will have an electrode spacing D that will range from 1 mm to 10 mm Standard cuvettes are widely available in 1 mm 2 mm and 4 mm spacing To obtain the required electric field
65. s 2 4 kV cm 10 s Serpersu 1985 3T3 fibroblasts 30 microns 1 2 1 5 kV cm 100 s Mir 1988 Murine fibroblasts 30 microns 1 4 2 kV cm 40 500 s Liang 1988 B lymphoblasts 15 microns 1 2 1 4 kV cm 100 s Press 1988 Poly morphonuclear leukocytes 30 microns 5 10 kV cm 1 5 s Hashimoto 1989 Yeast 5 microns 7 5 8 5 kV cm 50 s Bartoletti 1989 Fish eggs 200 microns 750 V cm 50 s Inoue 1990 Estimated cell diameter Actual diameter not mentioned in articles 6 2 PA 3000 Protocol Optimization 6 2 1 Choosing Starting Voltage and Pulse Width The most difficult initial decision in protocol optimization is selecting the starting voltage and pulse width There are several methods for doing this The simplest is to start with a published protocol For an example of one method we will start with a cell with a diameter of 20 microns and look up published values for that cell Our example cell will be CHO cells Chinese Hamster ovary cells Our goal is to transfect the cells with a plasmid containing a gene that we have inserted Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 6 2 PA 3000 Users Manual The first step is to determine your electroporation protocol needs Above is a form that may help you define those goals and to develop a starting point for optimization From the table above we see that Zerbib et al used a rectangular wave pulse of 1 5k volts per centimeter an
66. t permeate the cell That means that the first pulse must be above the cell electroporation threshold In PulseAgile protocols this pulse may have a shorter duration than published parameters because the first pulse does not have to do all of the work of inducing poration and transport simultaneously Second and subsequent pulses are used to increase effective pore area and to assist in molecular transport In general the area of the cells that is permeable during electroporation is proportional to the strength of the applied electric field The size of pores induced is roughly proportional to the width of the applied pulse According to one theory pores are formed in cells by a rearrangement of phospholipids to a transiently stable pore shape This rearrangement occurs normally in cells at a very low rate The applied electric field serves to increase the probability of formation of transiently Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 2 6 PA 3000 User Manual stable pores There is an energy hill that pores must climb before rearranging from transiently stable pores to normal bilipid layer cell membrane Therefore pores close at a slower rate than they form Thus for a brief time up to seconds a significant number of pores exist after the pulsed electric field is turned off This is the time during which electrophoretic pulses can work to move charged molecules such as DNA into cells For practica
67. tage Monitor this BNC connector is located at the bottom center of the back panel A coaxial cable is connected from this connector to oscilloscope Channel 1 As stated this replica is calibrated into 50 ohms The amplitude of the signal is 1 200 of the actual high voltage pulse That is a 1000 volt pulse will appear as a 5 0 volt pulse into 50 ohms at the oscilloscope To calculate an estimate of the actual high voltage pulse Pulse Amplitude Estimate Pulse Voltage Monitor in volts x 200 volts volt The pulse width and interval are the same as the high voltage pulse The pulse rise time out of this monitor is slower than the actual pulse rise time If pulse rise time measurements are critical than an external high voltage probe should be used see below external measurements Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 B 1 PA 3000 Users Manual Connection 3 Pulse Current Monitor this BNC connector is located at the bottom left of the back panel A coaxial cable is connected from this connector to oscilloscope channel 2 As stated this replica is calibrated into 50 ohms The amplitude of the signal is 1 20 of the actual pulse current resulting from the high voltage pulse That is pulse current of 100 Amps will appear as a 5 0 volt pulse into 50 ohms To calculate an estimate of the pulse current Pulse Current Estimate Pulse Current Monitor in volts x 20 Amps volt The pulse width and interv
68. tions There are two main cuvette related factors that influence the applied starting voltage cuvette electrode gap and the resistance of the electroporation medium Cuvette with 2 mm gap If in our example we chose to use PBS or similar ionic medium and a cuvette gap of two mm the following calculations would determine the applied voltage The resistivity of PBS is 60 cm We will fill the cuvette to a volume of 180 l The resistance of the solution is calculated by R l a R 60 cm 0 2 cm 0 9 cm2 R 13 The voltage needed across the cuvette is V Field strength X cuvette width V 1500 V cm X 0 2 cm V 300 volts From figure 3 3 on page 3 3 the actual voltage delivered at 13 is 84 of the set voltage Therefore the actual set voltage needs to be 300 0 84 357 volts Cuvette with a 4 mm gap The above calculation applied to a 4 mm cuvette with the same medium shows that a greater percent of the set voltage will be applied to the solution We will fill the cuvette to a volume of 400 l half full The resistance of the solution is calculated by r l a R 60 cm 0 4 cm 1 0 cm2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 6 4 PA 3000 Users Manual R 24 The voltage needed across the cuvette is V Field strength X cuvette width V 1500 V cm X 0 4 cm V 600 volts From the graph on page 3 3 the actual voltage delivered at 13 is 90
69. to estimate the actual pulse amplitude when the load resistance is low less than 100 ohms This is normally the case when ionic buffers such as PBS are used Press Enter to continue Step 18 Post pulsing options Push lt GO gt to replay _or lt ENTER gt to edit A Re run Protocol To run the same protocol again assuming the power was not turned off press the Go button The power supply charges to the voltage set in Group 1 B Edit Protocol If a change is desired press Enter and scroll through the protocol menu until the group to be modified is shown by default Edit Group 1 will show first Press Enter to modify the settings C Save Protocol If the protocol is to be saved either for later usage or for temporary storage while another protocol is developed first press the Enter button to return to the protocol menu then press Down in order for Save Protocol to be displayed Press the Enter button to go to the next screen gt gt Protocol Save lt lt Protocol 1 10 Scrolling up and down will reveal all protocol save slots Empty slots will have a space between the brackets on the right hand side and those that already contain data of a protocol will be marked with an asterisk between the brackets Select a protocol save slot and press the Enter button to save the current protocol to memory If a protocol save slot with previous data is selected the user is first asked to re confirm whether or not
70. to overwrite the existing data Please reference Appendix D System Operation Flowchart for more operation details Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 8 PA 3000 Users Manual Ch 5 5 5 System Check To verify proper system operation place a 4 mm spacing cuvette with 400 l PBS into the cuvette holder and close Enter a single 0 060 ms 60 s pulse with voltage set at 1000 volts and run The load estimate should be approximately 21 to 23 ohms at 25 oC If an oscilloscope is available the pulse voltage and current may be viewed Typical results are presented in Figure 5 3 and data log in Figure 5 4 1 gt 2 1 Ch 1 1 Volt 10 us 2 Ch 2 1 Volt 10 us Pulse Voltage Monitor 4 52 volts x 200 v v 904 volts Pulse Current Monitor 2 08 volts x 20 A v 41 6 Amps Resistance 904 41 6 22 ohms Figure 5 3 Scope V Mon and I Mon gt 06 08 99 3 18 50 45 gt Group Number Width Int VMon gt 1 1 0 060 0 130 995 gt Estimated load 22 ohms gt Estimated conductance 0 045 siemens gt Pulse Amplitude Approx 92 of PS Voltage gt Normal Completion Figure 5 4 Data Log Example Cyto pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 9 PA 3000 Users Manual Ch 5 Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 5 10 PA 3000 Users Manual Chapter 6 Getting St
71. y time the holder is open the high voltage is disabled 3 2 Very Important Concepts There are five very important concepts that the user needs to be familiar with to be able to use and properly interpret the readings provided by the PA 3000 electroporator They are 1 Load 2 Relationship between Power Supply Voltage and Pulse Amplitude 3 Pre Pulse Load Estimator 4 Pulse Droop 5 Aqueous Solution Heating Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 715 0990 3 1 PA 3000 User Manual 3 2 1 Load The tutorials in Chapter 2 explained that applying a voltage across a cuvette produces an electric field As a result of this application of electric field current electrons will flow through the solution in the cuvette If the solution is very ionic such as Phosphate Buffered Saline PBS it will be very conductive that is have a low resistance From Ohms Law this current is related to voltage by Current amperes Voltage volts Resistance ohms Also in common use is the term conductance which is Conductance siemens 1 Resistance The PA 3000 will estimate resistance conductance and present both on the log report generated after each protocol run If the resistance is too low the electroporator will automatically reset 3 2 2 Relationship Between Power Supply Voltage and Pulse Voltage When the user sets a power supply voltage via the User Interface that voltage is not the actual volt

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