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1. 2 1 2 2 2 4 2 5 2 6 2 7 2 8 3 1 3 3 3 4 3 5 4 1 4 2 4 3 5 1 5 2 5 4 B 1 B 2 B 4 B 5 vi List of Figures Electropores Pore Area Electric Field in a Cuvette Electric Field vs Cuvette Spacing Exponential Decay Generator Pulse Amplitude vs Time for an Exponential Decay Generator Uptake Calcein Cell Viability Log Report Example Power Supply and Pulse Amplitude Relationship Typical Relationship between Power Supply and Pulse Amplitude Minimum Pulse Interval for Decreasing Voltage between Pulses Minimum Pulse Interval for Increasing Voltage between Pulses PA 4000 Front Panel Features PA 4000 Back Panel Features PulseAgile Opening Screen showing current system status PulseAgile Interface Electroporation Mode Screen A Five Pulse Protocol Divided into Four Groups of Pulses of Varying Parameters The Last Protocol Log for the Basic Test Run The Pulse Train Delivered by the Basic Protocol Oscilloscope Output Example Simplified Circuit Diagram of Monitors Error in Voltage Monitor Due to Current Viewing Resistor Typical Pulse Voltage as a Percent of Power Supply Voltage Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 page 2 1 2 1 2 3 2 3 2 4 2 5 2 14 2 14 3 2 3 4 3 5 3 5 4 1 4 3 4 7 5 2 5 6 5 9 5 9 B 3 B 3 B 5 PA4000 User Manual rev 1 1 05 Caution Notice This instrument contains a high voltage power supply adjustable b
2. Four Parameter PulseAgile Electroporation System User Manual Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 1 410 787 1890 1 410 787 1891 FAX www cytopulse com Cyto Pulse Sciences Inc makes no warranty with respect to the product except for the warranty set forth in this Users Manual on page 7 1 The LIMITED WARRANTY SET FORTH ON PAGE 7 1 IS EXCLUSIVE AND NO OTHER WARRANTY WHETHER WRITTEN OR ORAL IS EXPRESSED OR IMPLIED Cyto Pulse Sciences Inc SPECIFICALLY DISCLAIMS IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE License Agreement PA 4000 Pulse Generator Cyto Pulse Sciences Inc Licensor conveys to the licensee for fee paid a nonexclusive nontransferable license to use the PulseAgile hardware and software equipment for research purposes into perpetuity Cyto Pulse Sciences Inc has patent allowance and patents pending covering the PulseAgile process If the licensee wishes to use the hardware and software for production or commercial purposes an additional license shall be required The equipment is not approved by the FDA for use in vitro or for in vivo diagnostics or therapy The information in this manual is subject to change without notice Copyright 2000 2005 Cyto Pulse Sciences Inc PA4000UMANrev 1 1 05 Price 100 00 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 i PA4000 User Manual rev 1 1 05 BlankPage ii Cyto Puls
3. 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 151 982 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 410 787 1890 6 5 PA 4000 User Manual Ch6 rev 1 1 05 Blank Page 6 6 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch7 rev 1 1 05 7 Customer 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 warranti
4. the system Stop Reset button and the indicator light emitting diodes LEDs that display the equipment status Model PA 4000 Four Paramater PutseA gi amp Electroporation System E US Patents 6 910 613 and 6 078 430 I I Fault Conditions Cyto Pulse Sciences Inc Core Emas boai er Poser VOFF MYON Ofle Pas Status Power Switch f Reset Button Line Mains Power 0 i Figure 4 1 PA 4000 Front Panel Features Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 4 1 PA 4000 User Manual Ch4 Rev 1 1 05 4 2 2 Back Panel Connections Line Mains Power Cord The line mains cord supplied with a grounded IEC connector must be plugged into the back of the unit Figure 4 2 Detail 1 shows the location DO NOT CONNECT THE OTHER END OF THE LINE MAINS CORD INTO THE WALL UNTIL ALL INSTALLATION STEPS ARE COMPLETE Serial Cable the supplied serial cable must be connected between the D Subminiature 9 position DB9 connector on the back of the unit and a computer This is the communication link between the microprocessor in the PA 4000 and the computer This is a standard RS 232 serial communication interface See Figure 4 2 Detail 2 Cuvette Interlock Cable The interlock cable with the RCA type phono jack from the Cuvette Holder must be connected to the jack labeled Cuvette Interlock at the top of the back panel See Figure 4 2 Detail 3 High Voltage Cable
5. 0 200 400 600 800 1000 Pulse Amplitude volts where p resistivity onm cm D plate spacing cm A plate contact area cm Figure 2 4 Electric Field vs Cuvette Spacing Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 3 PA 4000 User Manual Ch2 Rev 1 1 05 Additionally conductive properties are also described as conductivity Conductivity is simply the reciprocal of resistivity Conductivity 0 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 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 cm The same sized cuvette filled to different volumes results 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 p Resistance Q cm Q Cuvette and 1 mm 2 mm 4 mm Volume with 50 ul with 200 ul with 800 ul PBS 60 25 C 12 12 12 Distilled Water 18x10 3 6x10 3 6x10 3 6x10 1 Sigma PBS
6. 1890 PA4000 User Manual Ch1 rev 1 1 05 PA 201S Programmable Pulse Switch Option to PA 4000S restricted license sale A user programmable switch that allows the PA 4000 to drive up to eight electrode elements independently This add on system includes 1 PA 201 Programmable Pulse Switch 8 Outputs 1 PA 201UMAN PA 201User Manual 1 CS OPT Interface Cable Set DB25 cable HV cable 1 CS 201 Eight Wire Cable with plug for electrode connections PA 301S Pulse Booster Option to PA 4000S restricted license sale Capable of producing pulses up to 3000V for higher electric field applications This add on system includes 1 PA 301 Pulse Booster 3 1 Step Up Transformer 1 PA 301UMAN PA 301User Manual 1 CS OPT Interface Cable Set DB25 cable HV cable Optional Components Purchased Separately DS 100 A Tektronix digital oscilloscope with the CS DS cable set and software CS DS Oscilloscope Cable Set Three RG 58 coaxial cables with 50 ohm terminations PCL Laptop computer with the PulseAgile interface software installed Cuvettes Electrodes Plates and Holders CUV 01 Standard Cuvette 1 mm spacing one time use CUV 02 Standard Cuvette 2 mm spacing one time use CUV 04 Standard Cuvette 4 mm spacing one time use CUV M Multi pack five each of the above one time use CE 20 Cuvette Holder for standard cuvettes CE 24 Cuvette Holder for reversing electric fields in a cuvette for use with PA 201 TE 5 10 Tweezer Electrode 5 mm x
7. again This property will limit protocols where multiple pulses are required 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 is turned on only for the desired pulse duration This system still has a Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 5 PA 4000 User Manual Ch2 Rev 1 1 05 reservoir capacitor but it is only discharged by a few percent during a pulse 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 Together these properties provide a more repeatable pulsed electric field 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 Sciences PA 4000 PulseAgile Electroporator The PA 4000 PulseAgile electroporator provides the highest level of sophistication in the market today It provides control of all pulse parameters with the ability to set pulse width amplitude time between pulses and the electric field direction The PA 4000 system provides resea
8. clear vs blue cells Draw a graph of total cells vs blue cells Since this test does not differentiate between dead porated cells and living porated cells it serves as a first approximation The mid range voltage for the rest of our optimization studies will be a voltage that induces less than 50 blue cells The exact value to choose will depend upon your electroporation goals For this example we will chose the voltage that yields 25 blue cells At this value we know that at least 25 cell viability is maintained and that effective poration can be measured For this example we will assume that 500 volts not an actual measurement yields 25 blue cells 6 22 Amplitude of Low Voltage Pulses Since our goal for this electroporation protocol is to transfect CHO cells we will use a two voltage protocol since we know that it will be more efficient than a one voltage protocol Low voltage pulses move DNA into cells by electrophoresis and as far as we know do not porate cells For this example let s choose arbitrary parameters for the low voltage pulses such as 200 V cm or about 90 volts and long durations wide pulses of 2 ms Again as an arbitrary starting number we will apply 6 low voltage pulses 6 2 3 Optimization of First Pulse Two measurements need to be made for each pulse protocol tested One measurement needs to be a measurement of transfection efficiency The other measurement needs to be a measurement of cell viability Si
9. contact the factory for assistance Phone 410 787 1890 Internet www cytopulse com 4 6 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 PA 4000 User Manual Ch4 Rev 1 1 05 PA 4000 Interface Version 2 01 JC x File Tools Settings Help Options Connected EET SRE URN irrar r a System Jk CommLink Jk Electrode Holder High Voltage ji e Status Local Remote i mot Monitors Power Supply Voltmeter 5 volts Load Ohmmeter ohms Figure 4 3 PulseAgile Opening Screen showing current system status Protocol File PA 4000TEST PRO 12 14 2004 3 15 51 24 Mode Electroporation gt GRP NUM WIDTH INTVL SetV MonV gt 1 1 0 020 0 50 50 50 gt Estimated load gt 999 ohms gt Estimated conductance lt 0 001 siemens Figure 4 4 PA 4000Test pro Protocol Log Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 4 7 PA 4000 User Manual Ch4 Rev 1 1 05 4 7 Oscilloscope Installation Optional This installation procedure is for the digital oscilloscope option ordered from Cyto Pulse The scope package ordered from Cyto Pulse is composed of a 2 channel Tektronix digital storage oscilloscope and a set of three coaxial cables with 50 ohm terminations 1 Place the scope on top of or beside the PA 4000 2 Connect one coaxial cable from Ch1 of the scope to the Pulse Voltage Monitor BNC jack located at the bottom center of the back pane
10. cuvette plate spacing the cuvette plate area and the conductivity of the aqueous solution In tissue it is a function of the electrode spacing and the conductivity of the tissue The user may not know these In order to estimate the load so an estimate of pulse amplitude can be provided the PA 4000 uses a pre pulse generated by the computer before the high voltage is turned on and the protocol is started This pre pulse is 2 2 us in duration and 2 volts in amplitude The pulse is placed across the electrodes and the resulting current measured Since the voltage is known the resistance is calculated This is done by the microprocessor and presented at the bottom of log report as a resistance in ohms and a conductance in siemens see Figure 3 1 The pre pulse data is also used by the microprocessor to detect an output short or excessive conductivity of the ionic solution If the load estimate is less than 10 ohms the high voltage will not be enabled and an Output Shorted message will appear 3 1 6 Pulse Droop As explained in Section 2 2 3 rectangular wave electroporators also use storage or reservoir capacitors When the high voltage is turned on this capacitor is charged to an initial voltage by the internal power supply A pulse is generated when the capacitor is momentarily connected to the load and electrons in the capacitor are drained off like water running out of a reservoir After a set time the pulse width the capacitor is disconne
11. first pulse must 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 stable pores There is an energy hill that pores must climb before rearranging from 2 6 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 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 suc
12. formation can make the cell more resistant to detrimental effects of excessive pore expansion Roughness of the cell due to cell processes or villi is another example Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 9 PA 4000 User Manual Ch2 Rev 1 1 05 2 3 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 C 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 C and brought rapidly to 4 C These methods of prolonging pore life are rarely practical 2 3 10 Addition of Reagents Electroporation efficiency is much higher if the molecules that you want to introduce into cells DNA proteins and small molecules are in the cell suspension 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 4 Method Development Many combinations of pulse parameters are possible using PulseAgile electroporation Also there are several ways to arrive at an
13. in Section 5 3 Pulses Displays the number of pulses in the active group The number can be changed by directly typing in the number or by using the up and down arrows shown on the screen Pulse Amplitude Displays the reservoir capacitor charge voltage level setting of the active group The user sets the value within the allowed limits by typing in the number desired The unit is volts Range Two buttons to select High or Low which changes the range and resolution of the reservoir capacitor charge voltage Low Range allows from 4 to 400 volts in 2 volt increments High Range allows from 5 to 1100 volts in 5 volt increments Duration Displays the pulse duration width setting of the active group The user sets the value within the allowed limits by typing in the number desired The unit is milliseconds Since most pulse durations will likely be set in the microsecond range the duration will be frequently displayed in decimal values For example a display of 0 020milliseconds is 20 microseconds Interval Displays the pulse interval setting of the active group It is the amount of time from the beginning of one pulse to the beginning of the next pulse The unit is seconds The minimum value is 0 125 seconds Group List Displays the list of groups in the current protocol The groups will be executed in the order displayed There are three buttons to control the list Add or Alt A Used to add a group to the list Replace or
14. is pushed all the way in 3 22 Cuvette Holder Interlock If the Cuvette Holder handle is withdrawn from the plastic shield an interlock is tripped and the high voltage cannot be enabled A red Light Emitting Diode LED will illuminate on the front panel and Open will appear in the Electrode Holder status window The interlock is provided by a second cable that must be connected for the system to fully operate 3 2 3 Short Circuit Detection As described in Section 3 1 5 a load estimator circuit is used to determine the approximate resistance of the test sample before the high voltage is turned on If the value of resistance is too low below 10 ohms the PA 4000 software will display a message Output Shorted and will not turn on the high voltage or run the protocol 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 fully 3 24 Over Peak Current Limit Sensor The peak current sensor is used to detect excessive load current during a pulse If such a condition is detected the unit will shut down within a few microseconds and a red LED on the front panel called External Fault will be illuminated There are generally two causes of excessive load current e Arc in the Cuvette during a pulse If this fault occurs the cuvette or chamber must be examined to see what caused the fault and the situation corrected e Over heating the aqueous sol
15. obtain the required electric field intensities high voltage pulse generators have adjustable pulse E Voltage D amplitudes from tens of volts to over 1000 volts Figure 2 4 presents the electric Voltage field intensity for standard cuvettes and applied pulse voltages The concept of resistance is also Figure 2 3 Electric Field in a Cuvette 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 Q 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 14000 DE Ors mm 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 p rho This is given in ohm cm and is related to resistance by the formula Electric Field in Cuvette v cm resistance p D A
16. 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 4 2 Electroporation Medium Choice of the electroporation medium involves compromise Voltage drop during the pulse and heat generation are easily controlled when using high resistance low ionic medium The use of Cyto Pulse low conductivity medium is recommended for this purpose 2 4 3 Reporter Molecules Electroporation protocol development is much easier if a reporter molecule is available to readily assess the status of electroporation efficiency Some available materials are DNA with appropriate promoters lac Z B galactosidase green fluorescent protein Chloramphenicol acetyltransferase Luciferase antibiotic resistance Non DNA FITC labeled dextrans Calcein 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 mamma
17. on the reservoir capacitor PA 4000 at rest Displays an approximate measure of the pre pulse voltage used by the Load Estimator function in Low Range 2 volts in High Range 5 volts Load Ohmmeter Displays an estimate of the external load as determined by the Load Estimator function The estimate is displayed during the period from when the high voltage power supply is turned on to the finish of the protocol deilvery This value is then listed at the end of the Protocol Log Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 PA 4000 User Manual Ch5 rev 1 1 05 5 2 4 Last Protocol Log Window The window at the bottom center of the screen is the Last Protocol Log window The window displays the last protocol executed However all protocols run during a session are stored in memory At any time during a session the entire log history can be saved as a text file using the Log Save As tool described above 5 2 5 Electroporation Mode Control Panel Area The Electroporation Mode Control Panel Area in the center of the screen is where the pulse protocol parameters are set edited and reviewed The following is a description of each of the sections and input display boxes found in the Control Panel Group ID Displays the group number whose characteristics are shown in the rest of the Control Panel Area The Group ID is also shown highlighted in the Group List The Cyto Pulse Sciences concept of Pulse Groups is explained
18. optimal combination of electroporation parameters The following is one suggested methodology 1 Choose a starting point goals medium and reporter molecules 2 Optimize initial pore formation 3 Optimize follow up pulses 4 Optimize molecular transport 5 Repeat steps 2 3 4 if necessary and optimize other parameters if desired 2 4 1 Choosing a Starting Point First choose goals for the electroporation procedure The following questions may help e What molecules are you trying to get into the cell e What are the characteristics of the molecules size charge in solution etc e What type of cell are you using e What are the cells characteristics e What is the cell size e Do the cells have cell walls e Are there any substances in the proposed medium that are toxic to the cells e Is cell viability important e Is electroporation efficiency important e Are single clones to be selected from the cells e Are cells to be part of a library e Are cells to be used in bulk without cloning 2 10 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 e Will this protocol be used repeatedly or will this be a one time use e 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
19. pulse amplitude and the duration pulse width A method for doing this is to first calculate the minimum required applied electric field for the cell and compare it to published work on the cell in question or similar cell and confirm whether or not the published value is reasonable starting point 6 2 1 1 Calculate the Minimum Required Electric Field Our example cell is the CHO cell with an average diameter of 30 microns Our goal is to transfect the cells with a plasmid containing a gene that we have inserted Note 1 from the checklist gives V 1 5rE cos9 Solving for electric field E V 1 5r cos Assume V 1 volt and cos6 1 E 1 volt 1 5 15x10 cm 1 Therefore E 444 4 V cm This is the applied electric field that would produce the threshold voltage required to electroporate the average diameter cell at the poles nearest the electrodes Since all of the cells are not the same size and are not located near the electrodes higher electric field strength would be required to electroporate the maximum number of cells 6 2 1 2 Compare to Published Electric Field Data From the table above we see that Zerbib et al used a rectangular wave pulse that produced an electric field of 1500 V cm for a period of 50 microseconds in his electrode That field strength is over three times the threshold value Note that for any given cell size there is a 6 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4
20. the membrane at the specific site Since the breakdown voltage is approximately 1 volt the critical voltage for a cell in volts micron is E 1 1 5 r at the poles where cosB 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 micron or 333 volts cm In practice higher voltages are used since the above calculated voltage is only the minimum breakdown voltage The charge impressed upon a membrane during the application of a pulsed electric field creates a pressure across the cell membrane This pressure is an altered energy state around the membrane and creates a condition where pores can form Normally the most stable state of least energy for a membrane is a continuous bilipid layer membrane Another stable bilipid layer structure is an organized pore across the membrane This is a slightly higher energy state than a flat bilipid layer For a membrane to restructure from one of these stable states to another requires transitioning through a less organized higher energy state According to one model of pore formation pressure across a cell membrane created by charge redistribution reduces the transition energy and therefore makes it easier for pores to form Once a pore forms a path is created for electrical current to flow which relieves local pressure and maintains a favorable energy state for pores to remain ope
21. voltage pulses See section 3 1 5 to calculate temperature increase in an electroporation cuvette Another way to reduce heating is to use Cyto Pulse low conductivity medium 2 3 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 pulses 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 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 death using PulseAgile protocols 2 3 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
22. 000 User Manual Ch6 rev 1 1 05 wide range of fields used in the published work This variability is due to some extent to the differing balance of transfection efficiency and cell viability goals of the experimenters Soit would likely be reasonable to start with a pulsed electric field of 1500 V cm 6 2 1 3 Calculate the Starting Pulse Amplitude and PA 4000 Set Voltage The starting pulse amplitude will be influenced by two cuvette related factors the electrode gap and the resistance of the electroporation medium For this example assume that a standard cuvette with a gap of 2 mm and PBS or similar ionic medium will be used The following calculations determine the load resistance the pulse amplitude and the PA 4000 set voltage if the cuvette is filled to a volume of 180 uL From Chapter 2 the resistance of the solution is given by R p D A where p resistivity Q cm for PBS p 60Q cm D electrode gap cm A contact area cm Since A Volume D then R p D Volume Substituting R 60 Q cm 0 2cm 0 180cm gives R 13 30 Load Resistance The electric field desired in the cuvette is 1500 V cm and is given by E V D where V pulse amplitude volts So V E D V 1500 V cm 2cm V 300 volts Pulse Amplitude Recall that due to the PA 4000 s internal resistance the actual pulse voltage delivered to a 13 Q load is 84 of the set voltage see Figure 3 3 So the actual set voltage needs to be Vset Vpuls
23. 1 Load Resistance and Conductance 3 1 3 1 2 Power Supply Voltage Setting and Voltage Monitor 3 2 3 1 3 Relationship Between Power Supply and Pulse Amplitude 3 2 3 1 4 Changing Amplitude from Pulse to Pulse 3 4 3 1 4 1 Decreasing Voltage from One Pulse to the Next 3 4 3 1 4 2 Increasing Voltage from One Pulse to the Next 3 4 3 1 5 Pre Pulse Load Estimator 3 6 3 1 6 Pulse Droop 3 6 3 1 7 Aqueous Solution Heating 3 7 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 iii PA4000 User Manual rev 1 1 05 3 2 Safety Features 3 2 1 Cuvette Holder 3 2 2 Cuvette Holder Interlock 3 2 3 Short Circuit Detection 3 2 4 Over Peak Current Sensor 3 2 5 Over Average Current Sensor 3 2 6 Microprocessor Protection 3 2 7 Pulse Voltage and Current Monitors Set Up 4 1 Introduction 4 2 PA 4000 Pulse Generator 4 2 1 Front Panel Features 4 2 2 Back Panel Connections 4 3 Cuvette Holder 4 3 Computers 4 5 PulseAgile Software Installation 4 5 System Test 4 6 Oscilloscope Installation Optional Software Operation 5 1 5 2 5 3 Introduction The PulseAgile PA 4000 Interface Software 5 2 1 The Toolbar 5 2 1 1 File Pull down Menu 5 2 1 2 Tools Pull down Menu 5 2 1 3 Settings Pull down Menu 5 2 1 4 Help Pull down Menu 5 2 2 Tools Area 5 2 2 1 Mode Select Buttons 5 2 2 2 Tool Buttons 5 2 3 Status Area 5 2 3 1 Options Connected 5 2 3 2 System 5 2 3 3 Monitors 5 2 4 Last Protocol Log Window 5 2 5 Electroporation Mode Co
24. 10 mm pad TE 5R Tweezer Electrode 5 mm round pad NE 4 4 Parallel Row Needle Array 4 mm space 4 needles row NE 4 6 Parallel Row Needle Array 4 mm space 6 needles row NE 6 4 Parallel Row Needle Array 6 mm space 4 needles row NE 6 6 Parallel Row Needle Array 6 mm space 6 needles row FP C25 400 Coaxial Fusion Electrode 2 5 mm gap 350uL volume FP C25 800 Coaxial Fusion Electrode 2 5 mm gap 750uL volume FP C20 1000 Coaxial Fusion Electrode 2 mm gap 1000uL volume FE 10 Coaxial electrode holder for use with PA 101 96W A Array Electrode for PA 96W 96W P Microplate 96W PS Microplate Sterile y irradiated All of the products sold or licensed by Cyto Pulse Sciences Inc are for research use only and are not approved by the United Sates Food and Drug Administration for in vitro or in vivo diagnostics therapy or any other purpose Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 1 3 PA4000 User Manual Ch1 rev 1 1 05 Blank Page 1 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 2 Tutorials This chapter presents tutorials on electroporation and the various types of equipment used in the electroporation field 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 ele
25. 3 0 100 Voltage Load Estimator Result gt Estimated load gt 19 ohms Calculated Conductance gt Estimated conductance lt 0 053 siemens System Message gt Normal Completion Figure 5 3 The Last Protocol Log for the Basic Test Run session that has occurred since the software was started It can be viewed by clicking the Show Log History button The Log History may then be saved as a txt file by clicking File gt Log Save As or the Notebook button The Log may be printed to file or by a printer by selecting the Printer button Pulse 1 Pulse 2 Vv Pulse 3 Pulse 4 Pulse 5 Group 1 I Group 2 d l time Figure 5 4 The Pulse Train Delivered by the Basic Protocol Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 5 9 PA 4000 User Manual Ch5 rev 1 1 05 5 3 4 File Management File management conforms to standard Windows conventions The protocols are saved to and retrieved from a folder created at the time of software installation or to any user created folder The file extension used for a protocol file is pro The software automatically applies this extension when a protocol is saved Log History files are saved as plain text and are given the txt extension 5 10 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 PA 4000 User Manual Ch6 rev 1 1 05 6 Getting Started with PulseAgile Electroporation 6 1 Introduction This chapter provides
26. Alt P Used to change data within a group Changes made to values within a group will not take effect until Replace has been selected If a pulse parameter is changed but not Replaced an error dialog box will appear if the user tries to run the protocol The user will be prompted to replace the values first Remove or Alt M Deletes a group If a group is deleted the group numbers below the deleted group if any are renumbered accordingly Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 5 5 PA 4000 User Manual Ch5 rev 1 1 05 5 2 6 Running a protocol The following function buttons are used to run the current protocol e Turn HV ON or Alt H After all of the electroporation parameters are set and the user is ready start the protocol cuvette in place safety interlock engaged then click Turn HV ON The internal high voltage power supply HVPS turns on and charges the reservoir capacitor to the level set in Group 1 After seven seconds the Start button is highlighted and the system is ready to deliver pulses e Start or Alt S Clicking Start begins delivery of the protocol to the test sample The pulse groups are executed in sequential order A double beep signals the end of protocol execution if sounds were enabled If the Start button is not clicked within 80 seconds after turning on the HVPS the PA 4000 system will automatically reset itself e Reset or Alt R Clicking Reset stops the capa
27. An estimate of Rex is given in the Ohmmeter window Status area of the PulseAgile software interface and at the bottom of the Protocol Log report The above computation is performed by the PulseAgile software and also presented in the log report The number presented is the ratio of pulse voltage to power supply voltage As the External Load Resistance gets lower more voltage appears across the internal resistance and less voltage appears across the External Load This is shown in the graph in Figure B 4 This ratio estimate is accurate to about 10 The Rex estimate should not be used for precise analysis Using External Equipment to Measure Pulse Voltage and Current B 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA4000 User Manual AppB rev 1 1 05 The most accurate method to measure pulse voltage and pulse current is with an external 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 the 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 th
28. 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 which usually has a measurement accuracy of 3 to 4 Also described are the internal circuits of the monitors and how the scale factors are derived A Tektronix digital oscilloscope may be purchased from Cyto Pulse How to Use the Internal Monitors The PA 2000 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 oscilloscopes that have that option or by using an external 50 ohm coaxial termination A kit containing three 1 meter coaxial cables and three 50 ohm attenuators may be purchased from Cyto Pulse 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 volta
29. IDE THE HANDLE FORWARD UNTIL IT STOPS DO NOT CLOSE THE HANDLE WITHOUT A CUVETTE INSTALLED 4 4 Computers Place a computer next to the PA 4000 Ideally the computer will be equipped with an RS 232 serial port with a DB9 connector Connect the serial cable provided with the system between the serial ports on the PA 4000 and the computer If only USB ports are available on the computer then a USB to Serial converter will be necessary These converters are widely available however they are not available from Cyto Pulse Cyto Pulse Sciences makes no claim that the use of any USB to Serial converter is 100 applicable to the functionality of a PA 4000 Electroporation system The use of a RS 232 serial interface is recommended Use the manufacturer s instructions to install the converter first then connect the serial cable between the PA 4000 and the converter and proceed 4 5 PulseAgile Interface Software Installation The PulseAgile PA 4000 interface software must be run under Windows 95 or higher 32 bit If the software needs to be installed use the following procedure 1 Close all other programs and insert the CD ROM 2 Click Start from the Windows lower menu bar 3 Select Run which will display a dialog box 4 Type CDRomDriveDesignation setup then click OK 5 The setup program will begin follow the instructions from this point 4 6 System Test The following is a test of the basic system PA 4000 s
30. NA transfection may be different than that for other small soluble molecules It is known for instance that additional low voltage pulses enhance DNA transfection after the initial high voltage pulse See Chapter 2 for a further explanation 3 This value is important for highly conductive medium such as PBS or tissue culture medium Ifthe medium is highly conductive but the actual conductivity is unknown an approximation can be made by using the conductivity of PBS whose p 0 0333 siemens cm 4 Use the formula R p x cuvette gap cm sample volume cm with p Q cm 1 0 5 Use the formula V 2 x E x cuvette gap Yoappiied With E V cm and cuvette gap cm Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 6 1 PA 4000 User Manual Ch6 rev 1 1 05 Examples of several published protocols Cell B lymphoblasts 1 2 1 4 kV cm Press 1988 Polymorphonuclear 50 microns 5 10 kV cm 1 5 us Hashimoto 1989 leukocytes 75 85 Vien Bartoletti 1989 Fish eggs 750 V cm 50us Inoue 1990 Estimated cell diameter Actual diameter not mentioned in articles 6 2 An Example of Protocol Optimization Chapter 2 presented details regarding the factors that affect electroporation protocol optimization We will work through an example using Chinese Hamster Ovary CHO cells 6 2 1 Choose a Starting Pulse Voltage and Pulse Width The most difficult initial decisions are the choices of the starting
31. Protocols Running Protocols File Management to save and recall protocols Data Log Display printing and saving Status Display The following sections describe operation based on the above bullets Some things to remember There are three operating modes Electroporation Electrofusion and Programmable Pulse Switch Electrofusion mode may only be selected when a PA 101 is connected to the interface on the back panel Programmable Pulse Switch mode may only be selected when a PA 201 or PA 96W is connected to the interface on the back panel If an item is grayed out it cannot be accessed Either it is an option that is not installed or not available in the current mode Some commonly used functions have redundant control features i e they can be accessed from several places on the screen The default installation directory is C Program Files Pagile It will be different if you have selected another directory during installation The screen is divided into four areas within the main window The Title Bar and pulldown menus top the Tool buttons left the Control Panel for each operating mode center and the Status area right see Figure 5 1 When the cuvette holder is open The Electrode Holder window will display OPEN As a safety feature the high voltage power supply cannot be enabled and protocols cannot be run while the cuvette holder is open However protocols can be programmed and saved in this state Cyto P
32. TM Cyto Pulse Sciences Inc Model PA 4000 Four Parameter PulseAgile Electroporation System User Manual Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 1 410 787 1890 1 410 787 1891 FAX www cytopulse com Cyto Pulse Sciences Inc makes no warranty with respect to the product except for the warranty set forth in this Users Manual on page 7 1 The LIMITED WARRANTY SET FORTH ON PAGE 7 1 IS EXCLUSIVE AND NO OTHER WARRANTY WHETHER WRITTEN OR ORAL IS EXPRESSED OR IMPLIED Cyto Pulse Sciences Inc SPECIFICALLY DISCLAIMS IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE License Agreement PA 4000 Pulse Generator Cyto Pulse Sciences Inc Licensor conveys to the licensee for fee paid a nonexclusive nontransferable license to use the PulseAgile hardware and software equipment for research purposes into perpetuity Cyto Pulse Sciences Inc has patent allowance and patents pending covering the PulseAgile process If the licensee wishes to use the hardware and software for production or commercial purposes an additional license shall be required The equipment is not approved by the FDA for use in vitro or for in vivo diagnostics or therapy The information in this manual is subject to change without notice Copyright 2000 2005 Cyto Pulse Sciences Inc PA4000UMANrev 1 1 05 Price 100 00 FE Ce PA4000 User Manual rev 1 1 05 TM Cyto Pulse Sciences Inc Model PA 4000
33. age 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 4 8 Optimize Molecular Transport Molecular transport pulses are designed to move charged molecules into cells after pores have been induced The electric field of the material transport pulses is lower than the first pulses Values at or below threshold are used 2 4 9 Further Optimization of Molecular Transport Pulses All further optimization should focus on yield and cell viability simultaneously It is important to monitor both yield and cell viability in order 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 widths to be tested for each voltage Start with a range of 200 microseconds to 2 milliseconds Begin the optimization process using multiple pulses since multiple pulses will often be used in the final protocol Four pulses is a good starting point Keeping the number of pulses and pulse widths fixed test the effect of changing voltage within the range of voltages Then test the range of pulse widths against the optimal voltage Repeat this process until an optimum is found Again employing a factorial an
34. alysis 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 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 13 PA 4000 User Manual Ch2 Rev 1 1 05 Figure 2 7 shows that 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 us The PulseAgile protocols included a single pulse 3 3 kV cm 50 us followed 0 125 seconds later by 10 pulses of 1 ms and 0 4 kV cm with interval of either 0 125 s or 2 20 sec DU 145 prostate cancer cells were used at 2 X 10 cells ml in a 2 mm gap cuvette and 10 uM calcein Molecular transport and cell viability were calculated using calibrated flow cytometry with propidium iodide as the viability stain Figure 2 8 shows that cell viability was shown to not decrease with the PulseAgile protocols g 2 100 D A 2 amp aS gt 20 or Z x lt i 50 8 8 G o 0 0 Single Pulse PulseAgile Single Pulse PulseAgile Figure 2 7 Uptake Calcein Figure 2 8 Cell Viability Mark Prausnitz Ph D Georgia Institute of Technology provided the data for these g
35. ansfection efficiency or the best electroporation efficiency This manual is designed to help the user get the most benefit from using PulseAgile electroporation 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 4000 contains a high voltage power supply and was designed with safety features to protect both the user and the equipment Used properly the PA 4000 is a safe and reliable instrument Chapter 3 explains some important concepts related to safe and accurate use of the instrument Chapter 3 must be read and understood in order to properly set up this instrument Our goal is the safe and productive use of the PA 4000 This product shall only be used in the manner specified by the manufacturer Back Panel Symbols OA A 3 Protective Caution Caution Chassis Terminal Refer to Risk of Ground Conductor Documentation Electric Shock The PA 4000 including the optional PA 101 PA 201 PA 96W or PA 301 is rated for operation with line mains voltage of 100 240 VAC maximum current of 2 amps at 50 60 Hz The AC mains power supply cord is the disconnect device for this product The power supply cord shall be a Type SJT rated 300 Volts AC 18 AWG 105 C 3 conductor including ground This unit is rated for use at environmental conditions of 5 40 C maximum relative hum
36. cat D8662 2 Sigma water cat W3500 Resistivity is a strong function of temperature value given at 25 C If a 1000 volt pulse is applied to a cuvette with a 2 mm spacing and 200 ul 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 High Voltage switch remains closed until the capacitor is Power Supply completely discharged This capacitor is also called a reservoir capacitor The number of electrons that the capacitor can store size is measured in farads and given the symbol F which is the number of electrons per volt Figure 2 5 Exponential Decay Generator Charge Discharge Switch Resistance of Material in Solution Load Reservoir Capacitor 2 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 The pulse width is dependent on the size of the capacitor and
37. cceeding pulse is twice the width and half the amplitude The resistance and conductance estimates made before the protocol was run are shown at the bottom of the log Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 3 1 PA 4000 User Manual Ch3 Rev 1 1 05 Protocol File EXP HI PRO 12 3 2004 6 18 21 25 Mode Electroporation gt GRP NUM WIDTH INTVL SetV gt 1 1 0 020 1 000 1000 gt GRP NUM WIDTH INTVL SetV gt 2 1 0 040 1 000 500 gt GRP NUM WIDTH INTVL SetV gt 3 1 0 080 1 000 250 gt gt GRP NUM WIDTH INTVL SetV gt 4 1 0 0160 1 000 125 gt Estimated load 19 ohms gt Estimated conductance 0 053 siemens Figure3 1 Log Report Example 3 1 2 Power Supply Voltage Setting and Voltage Monitor There are two different power supply voltage numbers that appear in the log report shown in Figure 3 1 The first is the voltage set by the user using the PulseAgile interface software and is shown in the column labeled SetV This voltage setting is converted to digital words that the internal microprocessor uses to program the output voltage of the power supply The conversion is done in discrete levels or counts depending on the voltage range in use High Range 5 to 1100 volts 5 volts resolution Low Range 4 to 400 volts 2 volts resolution There are errors in these conversions and the final power supply voltage will be set to within 5 or two counts whichever is greater The second power
38. citor charging and or the delivery of pulses and can be used at any time e F12 Pushing F12 on the keyboard is the same as clicking Turn HV ON followed by Start Please note that the reservoir capacitor takes time to charge so there will be delay of approximately seven seconds before pulsing starts A double beep signals the end of protocol if sounds were enabled 5 3 Using the PulseAgile Interface Software This section will provide instruction regarding the conventions used to create Cyto Pulse PulseAgile protocols the setting up of a basic electroporation protocol and the data logging and file management features 5 3 1 Pulse Agile Protocol Conventions the Pulse Group PulseAgile electroporation allows pulse to pulse changes of parameters such as amplitude duration and interval In order to achieve this capability Cyto Pulse introduces the concept of the Pulse Group A Pulse Group is a set of pulses 1 to 99 in number of the same amplitude duration a k a pulse width and interval Figure 5 2 is a graphical representation of the concept Additionally the Pulse Group can contain other defined parameters specifically for PPS and electrofusion modes if those optional devices are in use Group 1 ile Group 2 oje Group 3 l Group 4 1 Pulse 1 Pulse 2 Pulses 1 Pulse Figure 5 2 A Five Pulse Protocol Divided into Four Groups of Pulses of Varying Parameters 5 6 Cyto Pulse Sciences Inc P O Box 609 Colu
39. confirm that the HV Off LED is illuminated to be sure that the protocol run has indeed completed before removing the cuvette from the holder Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 PA 4000 User Manual Ch5 rev 1 1 05 5 3 3 Reviewing the Last Protocol Log and Log History The Last Protocol Log is now ready for viewing and saving It provides a record of the protocol that was last delivered to the test sample A description of Last Protocol Log for this basic example is given in Figure 5 3 A representation of the actual pulse train is shown below in Figure 5 4 The parameters for every pulse delivered are recorded Note that the SetV and MonV parameters are referring to the voltage of the HVPS and reservoir capacitor not the pulse amplitude Each time the protocol is run the Last Protocol Log is refreshed and the previous results are added to the Log History The Log History is the record of the entire electroporation sac ee w Protocol File BASICTEST PRO ilename if applicable 47 2 4 Date MM DD YY DoW 12 T2004 6 18 51 35 Time 24 Hour Clock Mode Electroporation System Mode Delivered Pulse Parameters gt GRP NUM WIDTH subdivided by Group number gt 1 1 0 020 where 1 2 0 020 GRP Pulse Group z NUM Pulse in Group WIDTH Pulse Duration INTVL Time between gt GRP NUM WIDTH pulses gt 2 1 0 100 SetV Set HVPS Voltage gt 2 2 0 100 MonV Measured HVPS gt 2
40. cted from the load Long pulses allow more electrons to run out and the voltage decreases reservoir level drops just as in an exponential discharge pulser In rectangular wave electroporators the maximum pulse width is usually defined at the point that the pulse voltage at the end of the pulse is 95 of the initial voltage level This voltage drop is called droop and is determined by the size of the internal reservoir capacitor and the load resistance Droop is calculated by pulse width seconds C Rload Droop When using highly ionic loads the electrons are depleted faster Caution is required is setting pulse widths in these situations Typical pulse widths yielding 5 droop or less in the PA 4000 are Load Low Range High Range ohms 4 to 400 volts 5 to 1100 volts 10 420 us 150 us 20 840 us 300 us 40 1 68 ms 600 us 100 4 20 ms 1 50 ms 400 20 0 ms 6 00 ms 3 6 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 787 1890 PA 4000 User Manual Ch3 Rev 1 1 05 3 1 7 Aqueous Solution Heating Heating in the material being treated is a very important consideration The material is heated by energy from the pulses Energy is a function of many variables Energy in a single pulse is given by Pulse Amplitude Pulse Width watt seconds or joules Resistance of material Energy in one pulse The total energy in the pulse sequence used protocol is the sum of the en
41. ctropores Their presence allows molecules ions and water to 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 E cells can recover the electropores reseal spontaneously and cells will continue to grow and express their genetic material I A gt The use of electroporation became very popular through the 1980s because it was found to ER B be an exceptionally practical way to place drugs Nut 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 Figure 2 1 Electropores 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 r
42. e This means that it moves into the cell at the pole with the lower transmembrane voltage In theory pores could be induced in cell membranes with first pulses and the electric field reversed to move DNA into the cell on the side of greatest porosity That process would require a PA 201 Programmable Pulse Switch option 2 3 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 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 initial pulse widths in the range of 10 to 100 microseconds 2 3 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 voltage 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
43. e l appied Vset 300 V 0 84 Vset 357 volts PA 4000 Set Voltage If a4 mm cuvette were used with the same medium and filled to a volume of 400 ul half full about 90 of the set voltage would be applied to the solution So R 24Q 4 mm Load Resistance V 600 volts Pulse Amplitude Vset 667 volts PA 4000 Set Voltage Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 6 3 PA 4000 User Manual Ch6 rev 1 1 05 6 2 1 4 Quick Test of Starting Voltage Next we need a method to see if our starting voltage is reasonable Our starting values are e Cuvette chosen 4 mm gap cuvette e Medium or buffer PBS e Minimum Electric Field 444 V cm PA 4000 setting of 197 volts e Published Electric Field 1500 V cm PA 4000 setting of 667 Volts e Pulse width 50 us One quick method to test the range is to do a Trypan blue dye exclusion test This test evaluates total cells porated including dead cells Choose a range of voltages to test that includes the minimum voltage of 197 volts something above the highest published voltage 667 volts For instance perform single pulse protocols using the PA 4000 set from 200 volts to 700 volts in 50 volt increments The assay is performed by applying a 50 us duration pulse at a voltage within the chosen range to the CHO cell mixture in medium Immediately after the pulse is applied add an equal volume of commercial 0 4 Trypan blue dye Incubate for 5 minutes and count
44. e Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 PA4000 User Manual rev 1 1 05 Table of Contents page Introduction 1 1 PA 4000 PulseAgile Systems Options and Accessories 1 2 Tutorials 2 1 2 1 Electroporation 2 1 2 2 Electroporation Equipment 2 2 2 2 1 Electric Fields in Aqueous Solutions and Load 2 2 2 2 2 Exponential Decay ED Electroporators 2 4 2 2 3 Rectangular Wave Electroporators 2 6 2 2 4 Cyto Pulse Sciences PA 4000 PulseAgile Electroporator 2 6 2 3 Using and Optimizing PulseAgile Protocols 2 6 2 3 1 Background 2 7 2 3 2 Initial Pore Formation 2 7 2 3 3 Initial Pulse Width 2 8 2 3 4 Follow up High Voltage Pulse Further Pore Formation 2 8 2 3 5 Movement of Material into Cells 2 8 2 3 6 Cell Viability Factors Heat 2 9 2 3 7 Cell Viability Factors Excess Voltage 2 9 2 3 8 Other Cell Associated Factors 2 9 2 3 9 Solution Temperature Pore Closing Times 2 10 2 3 10 Addition of Reagents 2 10 2 4 Method Development 2 10 2 4 1 Choosing a Starting Point 2 10 2 4 2 Electroporation Medium 2 11 2 4 3 Reporter Molecules 2 11 2 4 4 Cell Viability 2 12 2 4 5 Electrical Parameters 2 12 2 4 5 1 Published Protocols 2 12 2 4 5 2 Cell Diameter 2 12 2 4 6 Optimize the first pulse 2 13 2 4 7 Optimize multiple high voltage pulses 2 13 2 4 8 Optimize molecular transport 2 13 2 4 9 Further optimization of Molecular Transport Pulses 2 13 2 5 References 2 15 Operational Concepts 3 1 3 1 Important Concepts 3 1 3 1
45. e 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 CA Load Resistance and Electric Field Predicting Model A Microsoft EXCEL model that will predict the external load is available from the Cyto Pulse This prediction is useful when setting up a protocol to determine the cuvette type to use the buffer type the volume to be treated and the electric field intensity in the cuvette Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 B 5 PA4000 User Manual AppC rev 1 1 05 Appendix C Declarations of Conformity CE Declaration of Conformity CB Test Certificate FCC Compliance CFR 47 Part 18 Subpart C Class A Equipment Industry Canada Compliance ICES 003 Category II Class A Equipment Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 C 1 PA4000 User Manual AppC rev 1 1 05 Blank Page C 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA4000 User Manual AppC re
46. easing Voltage from One Pulse to the Next To produce a pulse of one voltage followed by one of a higher voltage the reservoir capacitor voltage must first be increased i e additionally charged This voltage change takes time to occur and that time is related to the magnitude of the desired change The graph in Figure 3 5 gives the required time between pulses necessary in order for the pulse amplitude to be increased from one pulse to the next 3 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 787 1890 Minimum Pulse Interval seconds Minimum Pulse Interval milliseconds 300 0 200 0 100 0 0 0 PA 4000 User Manual Ch3 Rev 1 1 05 __ Low Range 4 to 400 volts N Minimum Pulse Interval Minimum PA 4000 Pulse Interval High Range 5 to 1100 volts Minimum Pulse Interval 0 01 0 1 1 Veurrent pulse Figure 3 4 Minimum Pulse Interval for Decreasing Voltage between Pulses Low Range 4 to 400 volts Minimum Pulse Interval 200 400 600 800 1000 1200 Voltage Change Vhext pulse Veurrent pulse Figure 3 5 Minimum Pulse Interval for Increasing Voltage between Pulses Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 3 5 PA 4000 User Manual Ch3 Rev 1 1 05 3 1 5 Pre Pulse Load Estimator The load is what the pulse voltage sees and it is a function of the material being electroporated In a cuvette this is a function of the
47. eciprocal of cell size If the upper limit threshold is reached lines 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 Total Pore Area A B C D Since the mechanism of electroporation is not well understood the development of protocols for a Pulse Amplitude x Pulse Duration particular application to a previously uncharacterized Initial Pulse cell or tissue have usually been achieved by empirically adjusting pulse parameters such as amplitude duration number and inter pulse interval Figure 2 2 Pore Area Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 1 PA 4000 User Manual Ch2 Rev 1 1 05 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
48. edium 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 3 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 the 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 The heat produced under the same condition is halved In practice multiple wide low voltage pulses are used to induce transport of material by electrophoresis after pores are formed by shorter high
49. ergy in all of the pulses For rectangular wave pulses the temperature increase in the material being treated is Neumann Sowers Jordan p66 Energy in all Pulses Cy S V Temperature Increase where Cp Specific heat joule gm K approximately 4 186 s specific mass gm cm approximately 1 v volume cm For example the resistance of 100 ul of PBS in a 2 mm cuvette is Resistance p spacing T 60Q cm od at Ty 240 For two pulses with amplitude 1000 volts and widths 100 us Total Energy 2 100 107 sec 8 3 joules and Temperatur e Increase Ess Joules 20 K 4 186 j gmK 1 gm I cm 100 ul 0 001cm gt ul NOTE The most effective method of monitoring temperature increases during the pulse is to use an oscilloscope to monitor the pulse current The pulse current should be constant over a pulse If the pulse current is increasing during the pulse then the material being treated is increasing in temperature during the pulse Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 3 7 PA 4000 User Manual Ch3 Rev 1 1 05 3 2 Safety Features There are many safety features designed into the equipment that will protect both the user and the PA 4000 This section will describe them 3 2 1 Cuvette Holder The Cuvette Holder was designed to prevent accidental contact with the high voltage pulse electrodes The electrodes can only apply voltage to the cuvettes when the handle
50. es 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 OF WARRANTY OR OTHER USES OF THIS PRODUCT CYTO PULSE S OBLIGATION TO REPAIR OR TO REPLACE TO THE EXTENT SET FORTH ABOVE CONSTITUTES THE EXCLUSIVE REMEDIES OF THE CUSTOMER FOR ANY BREACH OF WARRANTY This warranty shall not apply to products that 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 contact CYTO PULSE Customer Service through our website at www cytopulse com or phone 410 787 1890 or contact the local CYTO PULSE representative A determination if the product is still in warranty will be made If the warranty period is still in effect the user will be given an authorization number RMA to return the product If after receipt and inspection the product is found to be defective it will be replaced or repaired and returned to the customer If the product is found to have been modified or misused the user will be given a quote for repa
51. eyond 1 000 volts such voltage can be lethal The user must read this manual carefully before the instrument is placed into operation Removing the cover may void the warranty Do not connect or disconnect the high voltage cable with the high voltage enabled To connect or disconnect the cable turn line power off and unplug line cord Do not open the cuvette holder while the high voltage is on If a problem occurs during a run push the STOP RESET button on the front panel If there is any question about the operation of this instrument call Cyto Pulse Customer service Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 vii PA4000 User Manual rev 1 1 05 Blank Page viii Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 PA4000 User Manual Ch1 rev 1 1 05 1 Introduction Electroporation electropermeabilization has many uses in the fields of cell biology medicine and microbiology 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 PulseAgile technology gives research and medical scientists the tools needed for demanding new uses 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 tr
52. following are the calculated values In this example a 400 ul volume of PBS in a 4 mm cuvette at 19 C and power supply voltage of 1 000 volts was used Estimated Pulse Amplitude 2 24 div x 2 v div x 200 v v 896 volts Estimated Pulse Current 1 80 div x 1 v div x 20 A v 36 Amps Estimated PBS Resistance 896 v 36 A 24 9 ohms Estimated Total Charge 24 9 Ax 100 us 2 5 millicoulombs 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 2 below Figure B 1 Oscilloscope Output Example 1 Chi1 1 Volt 25 us 2 Ch2 500 mVolt 25 us B 2 Cyto Pulse 17 1890 PA4000 User Manual AppB rev 1 1 05 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 from High Voltage Power Supply and Capacitor T T T T T T T Tf Protection i i KS H E Resistors i i lt L E 2 2 ohms E Percent Brror External Load Buffer Solution Tissue Current Viewing ror Raste GVrent Viewing Resistor 0 20 ohms System Ground Earth Pulse Current Monitor a IMON Amplifier divide by 4 Pulse onge Monito
53. ge 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 Voltage 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 rise time measurements are critical than an external high voltage probe must be used see below external measurements 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
54. general guidelines to help with the building and optimizing of electroporation protocols Referring to published work is one way to start the electroporation protocol optimization process The following checklist may help Starting point for Electroporation Optimization 3 Published pulsed electric field If known See examples below V cm 4 Published pulse width If known See examples below 5 Published number of pulses If known See examples below Estimate the minimum electric field required to electroporate the cell lt 7 Desired transfection efficiency Helps define the endpoint for optimization 8 Desired cell viability Helps define the endpoint for optimization 9 Molecule for transfection DNA dye etc 10 What is the conductivity o of the medium siemens cm 11 Cuvette electrode gap 0 1 0 2 or 0 4 cm cm 12 Estimate the load resistance Q 13 Percent of voltage delivered to cuvette Use Figure 3 3 on page 3 4 applied 14 Calculate the necessary PA 4000 pulse voltage setting gt v 1 Use the formula V 1 5rE cos9 solving for E where V the minimum required threshold voltage across the cell Assume V 1 r the radius of the cell in cm E the strength of the applied electric field in V cm 8 the angle between the applied field direction and the normal vector of the membrane Assume cos6 1 for an electric field normal to the membrane 2 Optimization for D
55. gger signal precedes the pulse amplitude or current signals by a few hundred nanoseconds Connection to the trigger connector is not required A 50 ohm cable and termination at the oscilloscope should be used Hardware Option Interface this is the D Subminiature 25 position DB25 connector located at the top left of the back panel Figure 4 2 Detail 2 It is the control line over which the PA 4000 s internal microprocessor commands the optional equipment A shielded DB25 cable is supplied with the optional equipment This connector is the same type as that used for computer parallel interfaces However DO NOT USE THIS CONNECTOR TO CONNECT TO A COMPUTER PARALLEL PORT OR TO A PRINTER DAMAGE TO BOTH PIECES OF EQUIPMENT MAY OCCUR VOIDING THE PA 4000 WARRANTY 4 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 PA 4000 User Manual Ch4 Rev 1 1 05 PA 4000 Back Panel pea Detail 3 Detail 2 sseeebecsesccscsccscsees Detail 4 Detail 1 Detail 1 Line Power Fuse Holder Power Outlet for Use 2A 240VAC Cyto Pulse Optional Slo Blo Equipment that require Line Mains Power FOR USE WITH CYTO PULSE Plug Line Mains Cord here EQUIPMENT 100 240 VAC 50 60 Hz ONLY Detail 2 Interface Connection for Cyto Pulse Sciences Options ONLY No Printer or Parallel Port Connection Connect Serial Cable from Computer here Figure 4 2 PA 4000 Back Panel Features Cyto Pulse Science
56. h as DNA into cells For practical purposes after about 3 seconds the pores are closed to movement of large molecules into cells The optimization process should proceed iteratively modifying one variable at a time The following is a general outline for optimizing protocols 2 3 1 Background There are several components to PulseAgile protocols It helps to breakdown the optimization process into parts 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 3 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 0 2 to 2 volts 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 vector of
57. hey are 1 Adapting to existing protocols and optimizing from this starting point 2 Using cell diameter as a starting point 2 4 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 exponential decay protocols is as follows The first pulse is of the same voltage as the peak exponential voltage with a pulse width of 10 to 100 microseconds This pulse will be the pore forming pulse The second pulse is half the voltage and twice as wide The third pulse is half again the voltage and twice 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 2 4 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 es where r is the cell radius 2 12 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 2 4 6 Optimize the First Pulse There are many combinatio
58. idity 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 USA 410 787 1890 1 1 PA4000 User Manual Ch1 rev 1 1 05 PA 4000 PulseAgile Systems Options and Accessories The PA 4000 PulseAgile electroporation system is the base of a number of advanced component systems available from Cyto Pulse Sciences All of the systems include The PulseAgile interface software runs on a PC compatible desktop or laptop computer with the following minimum requirements 200 Mhz Intel Pentium Windows 95 CD ROM drive at least one serial port Cuvette Holder and Cuvettes The cuvette holder is designed for maximum safety of the user and integrity of the sample The cuvettes are available in 1mm 2mm 4mm gap spacing and individually sterile packaged Cyto Pulse System User Manual A User Manual is included for all systems and is written for use by life scientists The manual details the use and safety considerations of the system PA 4000S Advanced PulseAgile Rectangular Wave Electroporation System restricted license sale This system will run most published square wave protocols and offers the PulseAgile System parameter variations of pulse amplitude width and interval along with
59. ir 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 7 1 PA 4000 User Manual Ch7 rev 1 1 05 Blank page Appendix A Pulse Specifications Power Supply High Range Voltage Step Size Set Accuracy Low Range Voltage Step Size Set Accuracy Pulse Amplitude at 10 ohm load at 20 ohm load at 100 ohm load at 1000 ohm load Droop Pulse Over Current Shut Down Maximum Average Power Pulse Width Pulse Width Step Size Pulse Interval Pulse Interval Step Size Number of Groups Number of Pulses per Group Line Mains Power PA4000 User Manual AppA rev 1 1 05 5 to 1100 volts 5 volts 5 5 volts 4 to 400 volts 2 volt 5 2 volt 5 to 970 volts 5 to 1030 volts 5 to 1070 volts 5 to 1100 volts lt 5 at 20 ohms 150 us gt 125 amps gt 50 watts 1 us to 20 ms 1 us 0 125 to 400 sec 0 001 second 20 99 100 240 VAC 50 60 Hz IEC 320 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 A 1 PA4000 User Manual AppA rev 1 1 05 Blank Page A 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA4000 User Manual AppB rev 1 1 05 Appendix B Pulse Voltage and
60. l of the PA 4000 3 Connect one coaxial cable from Ch2 of the scope to the Pulse Current Monitor BNC jack bottom left of the back panel of the PA 4000 4 Connect one coaxial cable from EXT Trigger of the scope to the Pulse Trigger Out BNC connector located at the top right of the back panel of the PA 4000 Connect the scope line mains cord and turn power on Set up the scope as follows refer to the scope manual for assistance Push Ch1 Menu set Coupling DC BW Limit Off Volt Div Coarse Probe 1X Volts DIV knob 2 00V Vertical Position0 00 div using the position knob Push Ch2 Menu set Coupling DC BW Limit Off Volt Div Coarse Probe 1X Volts Div 2 00V Vertical Position4 00 div using the position knob Sec Div 25 us Push Trigger menu set Edge Slope Rising Source Ext Mode Normal Coupling DC Level 1 00V 4 8 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 PA 4000 User Manual Ch5 rev 1 1 05 5 Software Operation 5 1 Introduction This chapter will describe the various PulseAgile interface software features and functions The software conforms to standard Windows conventions and this manual assumes that the user is familiar with Microsoft Windows 95 or later The software performs the following functions Select operating mode Electroporation Electroporation with dielectrophoresis electrofusion Electroporation with programmable pulse switch Setting up
61. lian expression promoter makes an ideal reporter gene The fluorescent labeled dextrans are available in several molecular weights Proteins can be directly labeled with fluorescein Note that it is much harder to detect fluorescein labeled dextrans or proteins than it is to detect gene products because of the amplification inherent in DNA expression Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 11 PA 4000 User Manual Ch2 Rev 1 1 05 2 4 4 Cell Viability In addition to choosing a method for measuring yield a method for measuring cell viability is needed Methods include colony formation colony count before and after electroporation trypan blue dye uptake hours after the electroporation simple cell counts on tissue culture plates the day after electroporation KD 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 6 flow cytometric analysis or other fluorometric analyses of Calcein AM dye uptake 7 tritiated thymidine uptake There are many more methods although the gold standard is colony formation Note that vital dyes will penetrate permeabilized cells for some time after electroporation and cells that take up the dye may not be dead 2 4 5 Electrical Parameters There are at least two methods for choosing initial pulse parameters for electroporation protocols T
62. mbia MD 21045 410 715 0990 PA 4000 User Manual Ch5 rev 1 1 05 5 3 2 Setup and Run a Basic Protocol The following section is a guide to begin programming PulseAgile electroporation protocols It is meant as an exercise to familiarize the user with the functions and features of the PA 4000 system 5 3 2 1 A Basic PulseAgile Electroporation Protocol As an example the following pulse train will be programmed below and then run using the PulseAgile interface software Pulse 1 500 volts 20 us wait 0 2 seconds Pulse 2 500 volts 20 us wait 0 2 seconds Pulse 3 200 volts 100 us wait 0 3 seconds Pulse 4 200 volts 100 us wait 0 3 seconds Pulse 5 200 volts 100 us end 5 3 2 2 Program the Basic Electroporation Protocol Start the PulseAgile interface software Click the Electroporation mode select button When the mode is first selected there is no protocol loaded but default values are contained in the various input boxes Click the Add button above the Group Listto begin creating the protocol This action adds Groupi to the list Change the following parameters In Pulses type 2 In Pulse Amplitude type 500 In Pulse Duration type 0 020 In Interval type 0 200 Click the Replace button for the changes to take effect Group1 is now programmed Click the Add button This action adds Group2 is now added to the list Change the following parameters In Pulses type 3 In Pulse Amplitude type 200 In Pulse Duration t
63. n during application of the electric field Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 7 PA 4000 User Manual Ch2 Rev 1 1 05 Two practical conclusions derive from this model One is that continued application of high electric fields can force continued enlargement of pore size Thus electric fields cannot be applied indefinitely Another conclusion is that although pore formation is fast microseconds pore closure is slower milliseconds to seconds In practice pores are effectively closed by three seconds after application of pulsed electric fields even though some investigators have detected pores in cell membranes for more than 30 minutes Another factor to consider is that cells have a natural net charge across the cell membrane created by sodium pumps It is around 70 mV in most cells This charge is still present when an applied electric field re distributes charges within the cell The 70 mV them makes a negative charge that much larger and a positive charge that much smaller This results in a different transmembrane voltage at each pole of the cell in line with the electric field It is larger on the pole of the cell facing the negative electrode The larger transmembrane voltage results in a larger area of the cell membrane having a voltage greater than threshold and therefore a larger area containing pores The movement of DNA is toward the positive electrode since DNA has a net negative charg
64. narrower than the first pulse 2 3 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 of 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 2 8 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 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 voltage application that can be applied to cell suspensions Generally the most practical and effective molecular movement derived from electrophoresis is obtained when lower voltages are applied in multiple m
65. nce our hypothetical DNA molecule does not have an easily identified product we will choose a reporter molecule to determine transfection efficiency For this we will use the Green Fluorescent Protein reporter gene See the list of reporter molecules in Chapter 2 A positive transfection is recorded if cells are fluorescent under a fluorescent microscope 24 hours after returning to tissue culture Alternatively a flow cytometer can be used to measure fluorescence The percentage of positive cells is recorded The concentration of DNA may have to be optimized but we will use 10 ug ml as a starting point 6 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch6 rev 1 1 05 For cell viability we will use colony forming units This is done by plating a dilution of the cells onto a tissue culture plate allowing cells to adhere for four hours and overlaying the cells with agarose made with cell culture medium Any of the viability tests listed in Chapter 2 will do Since 500 Volts is our starting point from trypan blue experiments we will test the first pulses in 50 Volt increments ranging from 350 to 650 volts using a starting pulse width of 50 us Since two pulses are usually better than one pulse for the high voltage pulses we will use two initial pulses The first two pulses will be followed by six 90 volt pulses of 2 ms duration Plotting percent viability vs transfection efficiency gives us ou
66. nces Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 3 3 PA 4000 User Manual Ch3 Rev 1 1 05 1 000 2 Ko re e KA 0 960 ates Gl be 0 940 k Tier 4LEL LEL 0 920 0 900 d L L d a gi t r q T T P T q 0 880 EE E a 9 Hk dattrF eel eed ele AS oe Tec le iy ian a 0 860 0 840 Hd atte atrr Hd Stor TETT Pulse Amplitude Power Supply Voltage 0 820 JALE 44 PT ude dibi rif i a 0 800 10 100 1000 Load Resistance ohms Figure 3 3 Typical Relationship between Power Supply and Pulse Amplitude 3 1 4 Changing Pulse Amplitude from Pulse to Pulse One of the features of the PA 4000 is the ability to change the pulse amplitude up or down from one pulse to the next This ability is one element of PulseAgile electroporation It is important to note that there are limits to how fast a voltage change from pulse to pulse may be made 3 1 4 1 Decreasing Voltage from One Pulse to the Next To produce a pulse of one voltage followed by one of a lower voltage the reservoir capacitor voltage must first be decreased i e partially discharged This voltage change takes time to occur and that time is related to the magnitude of the desired change The graph in Figure 3 4 shows the minimum waiting time between pulses or Pulse Interval for this change to take place In High Range 125 ms is the minimum Pulse Interval for all changes 3 1 4 2 Incr
67. ns 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 electric field on cell viability Pick a range of electric fields to work with around the chosen starting electric field Generally twice the threshold voltage is a reasonable starting voltage A range of the starting voltage 33 50 should be sufficient Divide the range into equal parts of 25 50 volts cm and test the effect of each electric field on viability Pulse widths of 10 to 100 microseconds are 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 4 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 volt
68. ntrol Panel Area 5 2 6 Running a protocol Using the PulseAgile Interface Software 5 3 1 PulseAgile Protocol Conventions the Pulse Group 5 3 2 Setup and Run a Basic Protocol 5 3 2 1 A Basic PulseAgile Electroporation Protocol 5 3 2 2 Program the Basic Electroporation protocol 5 3 2 3 Save the Basic Protocol 5 3 2 4 Prepare a Test Sample and Run the Basic Protocol 5 3 3 Reviewing the Last Protocol Log and Log History 5 3 4 File Management Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 A WWWWWWwWW OO MOMMMOMC 4 5 4 8 ag q ND Oan hRRKRRWWWWWWPD TAAA O ONNNNOGO A oO PA4000 User Manual rev 1 1 05 6 Getting Started 6 1 6 1 Introduction 6 1 6 2 Example of Protocol Optimization 6 2 6 2 1 Choosing Starting Pulse Voltage and Pulse Width 6 2 6 2 1 1 Calculate the Minimum Required Electric Field 6 2 6 2 1 2 Compare to Published Electric Field Data 6 2 6 2 1 3 Calculate Starting Pulse Amplitude and PA 4000 Set Voltage 6 3 6 2 1 4 Quick Test of Starting Voltage 6 4 6 2 2 Amplitude of Low Voltage Pulses 6 4 6 2 3 Optimization of First Pulse 6 4 6 3 References 6 6 Ta Customer Service 7 1 7 1 Limited Warranty 7 1 7 2 Customer Service 7 1 Appendix A PA 4000 Datasheet Appendix B Pulse Voltage and Current Measurements Appendix C Declarations of Conformity Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 0990 V PA4000 User Manual rev 1 1 05
69. on and safety Asa result there are a number of protection and sensing circuits used to monitor the microprocessor operation to insure it is operating correctly The sensing circuits monitor proper power supply voltage proper cycle execution and adverse effects of sever line transients which could effect logic operation If any of these conditions are detected an UNIDENTIFIED FAULT window will appear in the operator interface To clear the fault the system must be reset by clicking OK in the operator window In some cases pushing the red Stop Reset button on the front panel is required 3 3 Pulse Voltage and Current Monitors All Cyto Pulse waveform generators have built in monitors to safely view replicas of the pulse amplitude and pulse current waveforms This permits the monitoring of these values for critical applications This method is the only precise way of determining the delivered pulse characteristics including load resistance and aqueous solution heating A digital oscilloscope and cable kit may be ordered from Cyto Pulse for this purpose It consists of three 50 ohm coaxial cables with three 50 ohm terminations The scale factors of the pulse monitors are Pulse Amplitude Pulse Voltage Monitor voltage X 200 into 50 ohms Pulse Current Pulse Current Monitor voltage X 20 into 50 ohms For example a 1000 volt pulse will produce a 5 volt replica at the connector A 100 amp pulse current will produce a 5 volt replica of the cu
70. on the left side of the screen and contains a set of frequently used function buttons These functions can also be accessed from the Toolbar as described above 5 2 2 1 Mode Select Buttons e Electroporation Button Selects the Electroporation mode e Electrofusion Button Selects the Electrofusion mode only if a PA 101 is connected e Electroporation Pro Pulse Switch Button Selects the Programmable Pulse Switch mode only if a PA 201 or a PA 96W is connected 5 2 2 2 Tool Buttons e Folder Button Opens a protocol e Disk Button Protocol Save As Saves a protocol under a user specified filename e Printer Button Prints the Last Protocol Log or the Log History user selectable to a file or printer e Calculator Button The Microsoft Windows Calculator e Notepad Button Saves the protocol Log History under a user specified filename e Handshake Button Communications Opens the Communications dialog box e Exit Button Exits the program Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 5 3 PA 4000 User Manual Ch5 rev 1 1 05 5 2 3 Status Area This area at the upper right corner of the screen displays up to date information about the system conditions 5 2 3 1 Options Connected This section shows which optional device is connected to the PA 4000 A check is shown in the appropriate box if a device is properly connected Only one optional device can be connected at a time 5 2 3 2 Sys
71. otocol Log History The user is prompted for a filename e Print Log Prints the Last Protocol Log or the Log History user selectable e Exit Exits the program 5 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 715 0990 PA 4000 User Manual Ch5 rev 1 1 05 5 2 1 2 Tools Pull down Menu e Calculator The Microsoft Windows calculator e Session History Opens a panel that allows the user to scroll through the Protocol Logs that have been run during the current session e Electroporation Selects the electroporation mode e Electrofusion Selects the Electrofusion mode only if a PA 101 is connected e Programmable Pulse Switch Selects the Programmable Pulse Switch mode only if a PA 201 or a PA 96W is connected e Quick Pulse Runs the open protocol without additional user input Pressing F12 achieves the same result The software activates Turn HV ONand START May be used to run the same protocol repeatedly A protocol must be open for this to work 5 2 1 3 Settings Pull down Menu e Communications This selection opens the communications dialog box Within the box are two functions 1 Port selection and 2 Communication link testing e Sound This selection turns the event beeper on and off e Reset Device Resets the system same as Reset button at top right 5 2 1 4 Help Pull down Menu e About Shows the Cyto Pulse logo software version and phone numbers 5 2 2 Tools Area The Tools area is found
72. protocol file and data logging management The system includes a b b i h be PA 4000 PA4 SW PA 4UMAN CE 20 CUV M CS L XX PulseAgile Advanced Rectangular Wave Electroporator PulseAgile Application Software PA 4000 User Manual Standard Cuvette Holder Cuvette Multi pack 5 each 1mm 2mm 4mm IEC Line Cord Serial Cable XX Country code Optional Cyto Pulse Add On Components PA 96W Programmable 96 Well Driver Option to PA 4000S restricted license sale Provides the ability to electroporate cells in selected wells of a 96 well microplate This add on system includes 1 a b b i oe PA 96W PA 96W UMAN 96W A 96W B 96W P CPS LCM C CS OPT Programmable 96 Well Driver PA 96W User Manual Electrode array for 96 Well Plate 96 Well Base 96 Well Polypropylene Plate square well flat bottom 10 each Low conductivity medium 500 ml bottle Interface Cable Set DB25 cable HV cable PA 101S Dielectrophoresis Option to PA4000S restricted license sale The sinewave AC generator dielectrophoresis option for cell alignment and fusion This add on system includes 1 a h b h 4 PA 101 PA 101UMAN FE 10 FE C25 400 CPS LCM C CS OPT AC Generator PA 101 User Manual Fusion Plate Holder Coaxial Electrodes 2 5 mm gap 350uL volume set of 3 Low conductivity medium 500 ml bottle Interface Cable Set DB25 cable HV cable Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787
73. r E VMON Amplifier unit gain Power Supply Back Panel Voltage Monitor t ter and data to computer and data log v itage Monitor Scale Factor 50 10 000 1 200 Current Monitor Scale Factor 0 20 4 1 20 HV Switch R a b Rext c Rext d Rext Figure B 2 Simplified Circuit Diagram of Monitors Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 B 3 PA4000 User Manual AppB rev 1 1 05 1 000 2 Ko re e KA 0 960 ates Gl be 0 940 k Tier 4LEL LEL 0 920 0 900 d L L d a gi t r q T T P T q 0 880 EE E a 9 Hk dattrF eel eed ele AS oe Tec le SET 0 860 0 840 Hd atte atrr Hd Stor TETT Pulse Amplitude Power Supply Voltage 0 820 JALE 44 PT ude dibi rif TIrFr 0 800 10 100 1000 Load Resistance ohms Figure B 4 Typical Relationship between Power Supply and Pulse Amplitude value of permitted External Resistance of 8 ohms Shown in Figure B 3 as the External Load Resistance gets larger the error gets smaller 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 RewRiia x Power Supply Voltage where Riotal Rinternal Rext Rinternal 2 5 ohms nominally This is a standard voltage divider relationship
74. r optimal first pulse voltage At this point our optimization gives us the desired efficiency and viability Also at this point a factorial analysis would be useful if the first optimization did not yield the desired results A 2 factorial analysis would allow simultaneous examination of voltage and pulse width of the two groups There are many paths to protocol optimization and this was one example 6 3 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
75. raphs 2 14 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 2 5 References 1 Sukharev S I Klenchin V A Serov S M Chernomordik L V and Chizmadzhev Y A Electroporation and electrophoretic DNA transfer into cells The effect of DNA interaction with electropores 1992 Biophys J 63 pp 1320 1327 Klenchin V A Sukharev S M Chernomordik L V Chizmadzhev Y A Electricaly induced DNA uptake by cells is a fast process involving DNA electrophoresis 1991 Biophys J 60 pp 804 811 Antonov P A Maximora V A Pancheva R P Heat shock and osmotically independent steps by DNA uptake after Escherichia coli electroporation Biochim Biophys Acta 1993 1216 2 pp 286 288 Sowers A E Mechanisms of electroporation and electrofusion in Guide to Electroporation and Electrofusion Editors Chang Chassy Saunders and Sowers 1992 Academic Press pp 119 138 Nickoloff Jac A ed 1995 Electroporation Protocols for Microorganisms Methods in Molecular Biology Volume 47 Humana Press Totowa New Jersey p 372 Nickoloff Jac A ed 1995 Animal Cell Electroporation and Electrofusion Protocols Methods in Molecular Biology Volume 48 Humana Press Totowa New Jersey p 369 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 Pre
76. rchers with the tools to design and implement optimal electroporation protocols The rest of this manual is devoted to the description and use of PulseAgile electroporation The goal is to provide you with the ability to get the best use of this patented technology 2 3 Using and Optimizing PulseAgile Protocols The simplest way to start using PulseAgile protocols is to begin with published pulse parameters for the cell type with which you are working Until PulseAgile protocol optimization is done standard published procedures and parameters can be used The 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 e Electrophoresis e Electroosmosis Thermodiffusion and osmotic flow of medium have also been proposed as transport mechanisms but there is little evidence that they play more than a minor role For any of these mechanisms to work the
77. rrent pulse An oscilloscope must be connected to the monitor ports on the back of the unit for viewing A complete description of the monitor circuits is given in Appendix B Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 3 9 PA 4000 User Manual Ch3 Rev 1 1 05 3 10 Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 787 1890 PA 4000 User Manual Ch4 Rev 1 1 05 4 Set Up NOTE THERE ARE SEVERAL IMPORTANT SAFETY AND OPERATIONAL CONCEPTS DESCRIBED IN CHAPTER 3 FOR YOUR SAFETY AND PROPER OPERATION OF THE PA 4000 CHAPTER 3 MUST BE READ FIRST 4 1 Introduction It is recommended to first setup only the basic system consisting of the PA 4000 a computer and the cuvette holder Test this configuration before connecting any optional equipment that may have been purchased The connections and set up for using the optional equipment PA 96W PA 101 PA 201 and PA 301 are described in their respective User Manuals If you purchased the Laptop Option the PulseAgile software was installed at the factory DO NOT PLUG IN THE LINE MAINS CORD UNTIL ALL OF THE SET UP PROCEDURES DESCRIBED BELOW HAVE BEEN COMPLETED 4 2 PA 4000 Pulse Generator 4 2 1 Front Panel Features Place the PA 4000 Pulse Generator on a tabletop There are no connections to be made to the pulse generator front panel shown in Figure 4 1 The three functions on the front panel are the Line Mains Power switch
78. s Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 4 3 PA 4000 User Manual Ch4 Rev 1 1 05 Detail 3 Cuvette Interlock Pulse Trigger Out Phono Jack BNC Connector Connector Connect optional oscilloscope trigger cable here Connect Cuvette Interlock Cable here Serial Number Label Detail 4 HV Out Monitors and System Ground High Voltage Connectors Use Banana Jacks OR MHV Jack Never use both at once 7 lack Never try to force a BNC onto the MHV jack Current Monito System Ground Stud f po 20A 10 32 bolt i Pulse Current Monitor Pulse Voltage Monitor 1V 20 Amps 1V 200 Volts Figure 4 2 cont d PA 4000 Back Panel Features 4 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 PA 4000 User Manual Ch4 Rev 1 1 05 4 3 The CE 20 Cuvette Holder The CE 20 cuvette holder will accept industry standard cuvettes The holder has two cable assemblies one for the pulsed high voltage connection and one for a safety interlock The larger diameter cable must be plugged into the Pulse Out MHV jack on the back panel of the PA 4000 This connector is the only one that will accept the high voltage cable The safety interlock cable must be plugged into the RCA type phono jack labeled Cuvette Interlock on the back panel of the PA 4000 The interlock must be satisfied for the system to properly operate PLACE AN EMPTY 4 MM CUVETTE IN THE CUVETTE HOLDER AND SL
79. s a cuvette produces an electric field As a result of this electric field current electrons will flow through the material contained between the plates of the cuvette The material is said to be presenting an electrical load on the system and will resist the flow of current to one extent or another From physics Ohms Law says this resistance is related to voltage and current by te voltage ar resistance current amperes If the material is very ionic such as Phosphate Buffered Saline PBS it will be very conductive i e it will have a low resistance If the material is tissue it will be less conductive than the PBS i e it will have a higher resistance and less current will flow Inversely conductance is given by slemens conductanc e ohms The PA 4000 will estimate resistance conductance and present both in the log report generated after each protocol run The number printed in the log is the estimate before the protocol is run Since the resistance of ionic solutions such as PBS is very sensitive to temperature the resistance will change decrease after each pulse due to heating The resistance estimate circuit operates between 10 ohms and 100 ohms Below 10 ohms the system will display a message Output Shorted and the high voltage will not be enabled Above 100 ohms the reading will be gt 700 ohms An example log report is given in Figure 3 1 Shown in the log is a PulseAgile protocol in which each su
80. ss Boca Raton pp 105 121 Chang D C Chassy B M Saunders J A and Sowers A E eds 1992 Guide to Electroporation and Electrofusion Academic press San Diego p 581 Dimitrov D S and Sowers A E 1990 Membrane electroporation fast molecular exchange by electroosmosis Biochimica et Biophysica Acta 1022 pp 381 392 10 Neuman E Sowers A E and Jordan C A eds 1989 Electroporation and Electrofusion in Cell Biology Plenum Press New York pp 581 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 15 PA 4000 User Manual Ch2 Rev 1 1 05 Blank Page 2 16 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch3 Rev 1 1 05 3 Operational Concepts This chapter describes some important concepts with respect to the proper and safe use of the PA 4000 electroporation system 3 1 Important Concepts There are seven important concepts with which the user needs to be familiar in order to be able to properly use and interpret the readings provided by the PA 4000 electroporator e Load Resistance and Conductance e Power Supply Voltage Monitor e Relationship between Power Supply Voltage and Pulse Amplitude e Changing Pulse Amplitude from pulse to pulse e Pre Pulse Load Estimator e Pulse Droop e Aqueous Solution Heating 3 1 1 Load Resistance and Conductance The tutorials in Chapter 2 explained that applying a voltage acros
81. supply voltage number is the value measured by an internal voltage monitor circuit at the internal reservoir capacitor This measurement is also converted into digital counts and is presented in the log report in the column labeled MonV Therefore the user can see the intended Power Supply Voltage SetV and the measured actual Power Supply Voltage MonV The measurements of the actual voltage and the digitizing processes used also have errors that are on the order of 5 or two counts whichever is greater 3 1 3 Relationship Between Power Supply Voltage and Pulse Amplitude When the user sets a power supply voltage in the PulseAgile Interface software that voltage will not be the voltage of the pulse that will appear across the material being treated cuvette tissue etc The actual pulse amplitude can be estimated if the value of the load aqueous solution or tissue resistance is known The circuit diagram in Figure 3 2 gives the reason for the difference 3 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 787 1890 PA 4000 User Manual Ch3 Rev 1 1 05 High Voltage Switch Pulse Amplitude at cuvette Rsource Internal Aqueous Rioaad Solution in Cuvette Figure 3 2 Power Supply Voltage and Pulse Amplitude Relationship As shown when the switch is closed the full Power Supply voltage actually appears across two resistances that inside the box and that outside the box i e aqueous solution in cu
82. t pulses may be shortened 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 um 55 v cm Mammalian Cell 50 um 200v cm Red Blood Cell 7 um 1430 v cm Bacterial Cell 1 um 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 2 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 4000 User Manual Ch2 Rev 1 1 05 In electroporation applications a lt Spacing gt 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
83. t area If TEST OK does not appear in the box then try choosing another COM port in the Port Selection area and repeating the test If communication can t be established then turn off the PA 4000 and check the serial cable connection to both the PA 4000 and the computer or USB to Serial adapter if applicable Once communication is established the test may proceed Then e Click on the file folder icon on the left side of the screen e Open the protocol file called PA 4000Test pro e Click Turn HV ON What should happen The protocol settings are downloaded to the PA 4000 s internal microprocessor The red Pulse LED will flash once indicating that the low voltage load estimator pulse has been sent to the cuvette holder Soon thereafter the red HV On LED will turn on and the High Voltage window should display On Then the red Off Zero LED will illuminate The high voltage power supply is now enabled and the reservoir capacitor is being charged After approximately seven seconds the START button is enabled The Power Supply Voltage window will display 50 volts and the Load Ohmmeter window will display 100 Ohms Then e Click START What should happen The PA 4000 runs the protocol A protocol log as shown in Figure 4 4 should appear in the Last Protocol Log window Again a more detailed description of the software features follows in Chapter 5 This completes the set up and testing of the basic PA 4000 system If there is a problem
84. tem This section shows the current PA 4000 system status These conditions frequently change during operation CommLink This box displays the status of the RS 232 serial communications link OK Communications is established and functional ERROR There is a problem with the communications link Electrode Holder This box displays whether or not the electrode holder in use is ready to receive pulses and the current status of the safety interlock OK The electrode holder is closed the safety interlock is satisfied OPEN The electrode holder is open the safety interlock is not satisfied High Voltage Displays the state of the internal high voltage power supply HVPS ON The power supply is on and the reservoir capacitor is charging OFF The power supply is off Status This box shows the current functional status of the PA 4000 It displays Ready The PA 4000 is ready to accept instructions to begin a protocol Download The computer is loading protocol to the internal microprocessor Charging The system is charging the reservoir capacitor to the set voltage Pulsing The protocol is being delivered to the electrode holder 5 2 3 3 Monitors Power Supply Voltage Displays the voltage of the reservoir capacitor not the pulse amplitude This display is operational at all times except when pulses are being delivered During charging Displays the voltage updated every second or so After charging Displays the final voltage
85. 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 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 B 1 PA4000 User Manual AppB rev 1 1 05 The pulse width and interval 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 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 rectangular wave pulse is calculated by PW Total Charge i t dt 0 Ie PW_incoulombs where 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 An example of an oscilloscope output is presented in Figure B 1 The top trace Channel 1 is the Voltage Monitor and the bottom trace Channel 2 is the Current Monitor The
86. the high voltage coaxial cable from the cuvette holder must be plugged into the Pulse Out MHV jack There is only one such jack on the back panel See Figure 4 2 Detail 4 The MHV connector is similar to the low voltage BNC connector however DO NOT ATTEMPT TO FORCE A BNC PLUG ONTO THE MHV JACK BOTH CONNECTORS CAN BE DAMAGED VOIDING THE PA 4000 WARRANTY 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 metal water pipe is satisfactory See Figure 4 2 Detail 4 Pulse Voltage Monitor Optional this BNC jack is available for the user who wishes to observe measure the pulse amplitude using an oscilloscope Figure 4 2 Detail 4 Connection to this jack is not required A 50 ohm termination at the oscilloscope is required A 50 ohm cable with terminations may be ordered from Cyto Pulse Pulse Current Monitor Optional this BNC jack is available for the user who wishes to observe measure the pulse current using an oscilloscope Figure 4 2 Detail 4 Connection to this jack is not required A 50 ohm termination at the oscilloscope is required A 50 ohm cable with terminations may be ordered from Cyto Pulse Pulse Trigger Out Optional this BNC jack is available for the researcher who wishes to observe measure the pulse signals using an oscilloscope Figure 4 2 Detail 3 It is connected to an oscilloscope trigger input The tri
87. 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 porator has a 500 uF reservoir capacitor and discharges into a 2 mm cuvette filled with 200 ul 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 uF 500 uF and 5000 uF reservoir capacitor and a 16 ohm Load Resistance The waveform follows a standard exponential or half time decay Voltage volts 0 i i i i eae 0 50 100 150 200 Time From Pulse Start milliseconds Figure 2 6 Pulse Amplitude vs Time for an Exponential Decay Waveform The Exponential Decay pulser although inexpensive is a relatively crude device As can be seen from the above example the amplitude needed for electroporating is in the early portion of the pulse but then the total area under the curve contributes to heating the sample Additionally the pulse width is dependent on the conductivity of the solution or tissue being porated and without compensation changes from one experiment to the next will cause the pulse width to change Finally since the capacitor is totally discharged in a single pulse it must be totally recharged before it can be used
88. time than simple diffusion For example 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 subsequen
89. ty should not pose a problem The internal high voltage power supply will turn on if the load current test is passed The system allows about seven seconds for the reservoir capacitor to charge On the front panel the HV ON LED illuminates followed shortly by the Off Zero LED The Off Zero LED turns on at the following conditions In Low Range for all voltage settings 4 400 Volts In High Range for voltage settings above 10 Volts 15 1100 Volts The Start button will be enabled when the reservoir capacitor is fully charged Click the Start button The programmed pulses are delivered to the cuvette beginning with Group1 The Pulse LED illuminates as each pulse is delivered The System Status box will display Pulsing A double beep signals the end of protocol execution if sounds are enabled Try Quick Pulse Pressing F12 on the keyboard will perform both functions of clicking Turn HV ON and Start If this option is used remember that there is a built in seven second charging delay before pulses are delivered When the protocol run is complete the high voltage power supply will be turned off and the reservoir capacitor will be discharged The system will return to the following status The HV OFF LED will be on The HV ON LED will be off The Off Zero LED will be off The System Status box will display Ready The Turn HV On button will be highlighted At the end of protocol execution it is a good safety practice to
90. ulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 5 1 PA 4000 User Manual Ch5 rev 1 1 05 5 2 The PulseAgile PA 4000 Interface Software Start the PulseAgile Interface software A screen will appear that shows standard Windows type pull down menus at the top a Tools area on the left and a Status area on the right shown in Figure 4 3 Now click the button labeled Electroporation which will add the Control Panel area and the Last Protocol Log window to the center of the display The screen should now appear as shown in Figure 5 1 PA 4000 Interface Version 2 01 File Tools Settings Help ELECTROPORATION T ME foo 00 Rest Options Connected Pi rerarar CommLink Jk Electrode Holder High Voltage fear Status Local Remote F f Monitors Power Supply Voltmeter 5 volts Load Ohmmeter ohms Interval 0 500 Range 6 High C Low Duration Replace fo 1 oo ms Remove Last Protocol Log a Figure 5 1 PulseAgile Interface Electroporation Mode Screen 5 2 1 The Toolbar Across the top left of the main window is a list of standard Windows pull down menus 5 2 1 1 File Pull down Menu e Open Opens a previously saved protocol e Save Saves the current protocol overwriting the file if it had been previously saved e Save Protocol As Saves the current protocol The user is prompted for a filename e Log Save As Saves the pr
91. ution in the cuvette This type of fault will occur if an ionic buffer such as PBS is used and or the user sets the pulse repetition rate too high Each pulse heats up the solution Heat decreases the resistance of the solution Eventually the resistance becomes so low that excessive current will flow In some cases the temperature increase can be so large that the aqueous solution in the cuvette boils Creating excessive heat must be avoided Cell lysis will occur long before the temperature reaches the boiling point Ina 1mm or 2mm cuvette this can happen in a few pulses at high voltage 3 2 5 Over Average Current Limit Sensor The average current sensor is used to detect excessive charging and discharging of the reservoir capacitor during complex protocol runs PulseAgile capability permits an infinite number of waveform combinations so it is not possible to describe each condition that will trigger this fault circuit In general protocols with a large number of repetitive pulses of very wide pulse widths and short pulse intervals can trigger this fault In such cases it is likely that substantial heating of the test sample would take place further enhancing the current draw and probably doing harm to the cells under treatment 3 8 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 410 787 1890 PA 4000 User Manual Ch3 Rev 1 1 05 3 2 6 Microprocessor Protection A microprocessor is used in the PA 4000 to control system operati
92. v 1 1 05 Replace with CE Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 C 3 PA4000 User Manual AppC rev 1 1 05 Blank Page C 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA4000 User Manual AppC rev 1 1 05 Replace with CB Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 C 5 PA4000 User Manual AppC rev 1 1 05 Blank Page C 6 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA4000 User Manual AppC rev 1 1 05 Replace with FCC Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 C 7 PA4000 User Manual AppC rev 1 1 05 Blank Page C 8 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA4000 User Manual AppC rev 1 1 05 Replace with Industry Canada Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 C 9 PA4000 User Manual AppC rev 1 1 05 Blank Page C 10 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890
93. vette or tissue The resistance inside the box called source resistance Rsource IS the inherent resistance in the high voltage switch and an additional resistance included to prevent excessive current from flowing if the output is inadvertently shorted The magnitude of the source resistance is usually a few ohms Again from Ohms Law Power Supply voltage R source AR toaa Total Current Amps From this relationship the voltage that appears across the load is always less than the power supply voltage If the power supply is set SetV to 400 volts the high voltage capacitor is charged to 400 volts If R source IS 2 ohms and R waa IS 18 ohms then the total current flowing from the high voltage capacitor through both resistances is 20 Amps The Pulse Amplitude is given by Pulse Amplitude volts Total Current R ma In the example above the pulse amplitude is 380 volts or 95 of SetV Another derived equation to calculate pulse amplitude is Ruaa Rra 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 shows the typical power supply voltage vs pulse amplitude relationship as a function of R waa If the load resistance is larger than 100 ohms than the difference between the power supply voltage and pulse amplitude voltage is less than 2 Pulse Amplitude Power Supply Voltage SetV Cyto Pulse Scie
94. ype 0 100 In Interval type 0 300 Click the Replace button Group2 is now programmed 5 3 2 3 Save the Basic Protocol At this point the protocol can be saved to disk if desired Follow these steps to save Click the Floppy Disk tool button or click File gt Protocol Save As Type a name for the file in the dialog box Click Save Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 5 7 PA 4000 User Manual Ch5 rev 1 1 05 5 3 2 4 Prepare a Test Sample and Run the Basic Protocol 5 8 Once the protocol has been setup it can be executed First place 400 uL of PBS in a 4mm cuvette An empty cuvette can be used as well but the Load Estimator result will be different than that discussed below Insert the cuvette in the CE 20 Cuvette Holder slide the handle forward until it is closed Check the System Status box to see that Ready is displayed Click the Turn HV ON button A low voltage approximately 2 5 volts pre pulse is delivered to the cuvette This pulse is used by the internal microprocessor to estimate the resistance of the load the aqueous solution in the cuvette Troubleshooting hint If the pre pulse current detected is too high the protocol run will cease An Output Shorted window will appear Click OK to acknowledge The solution in the cuvette must be checked for excessive conductivity before proceeding If the test cuvette was prepared exactly as described above the conductivi
95. ystem with no optional equipment attached using a preprogrammed protocol located in the default protocol folder A more detailed explanation of the use of the PulseAgile software is covered in Chapter 5 First e Plug in the Line Mains cord for the PA 4000 e Turn on the Line Mains power for the PA 4000 What should happen The green Power LED on the front panel will illuminate followed by the green HV Off LED This delay is due to system checks performed by the PA 4000 s internal microprocessor If the CE 20 handle has not been fully pushed in the red Cuvette Open LED will be illuminated If so slide it forward to engage the interlock and then the red Cuvette Open LED will turn off Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 Voice 410 787 1890 4 5 PA 4000 User Manual Ch4 Rev 1 1 05 Then e Start the PulseAgile PA 4000 Interface software What should happen The screen should appear like that as shown in Figure 4 3 The CommLink window should show OK This display indicates that the computer is communicating with the internal microprocessor of the PA 4000 Additionally the Electrode Holder window should display OK the High Voltage window should display OFF and the Status window should say Ready The Power Supply Voltage window will display 5 volts Troubleshooting tip If ERROR appears in the CommLink window then click Settings gt Communications on the upper toolbar Click Testin the Comms Tes

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