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1. Rev 1 11 04 Appendix C Electrode Array Cleaning Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 C 1 PA 96W User Manual Rev 1 11 04 Blank Page C 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 Cyto Pulse Electro poration and Electrofusion Systems for In vivo therapeutic delivery Gene therapy Immunotherapy Hybridoma production Vaccine delivery Nuclear transfer mRNA delivery siRNA delivery plasmid delivery Headquarters P O Box 609 Columbia MD 21045 USA Voice 410 71 5 0990 Fax 410 71 5 2148 Web veww cyto pulse com Mar 04 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 Cyto Pulse Sciences Inc en device and treatment development el P r ocedur e Cyto Pulse 96W A Electrode Array Cleaning Procedure Cleaning of 96 well array after use e Rinse with distilled water Immediately after use e Optionally the array may be cleaned using a mild detergent such as Windex e Rinse thoroughly in distilled water Disinfecting array for use If the array needs to be disinfected because of a biohazard the disinfecting or sterilizing procedures below may be used Consult with your Biosafety Officer for sterilizing procedures appropriate for specific biohazards e Soak array for 10 minutes in 4 Sodium Hydroxide Rinse with 70 Isopropanol Soak array for 10 minutes in 3 Hydrogen Peroxid
2. e Polynucleotide concentration e Medium formula Ultra Low Cost Per Well e Reusable electrode array e Low cost Polypropylene microplates Flexible Pulsing Protocols set from Windows Interface e All wells simultaneously One column at a time One Row at a time Uses e Proteomics and Functional Genomics a siRNA Inhibition gt Screen DNA libraries Antisense inhibition Intracellular monoclonal antibodies o Drug target identifier Cell specific responses o Gene function and regulation e Research HELA Transfection Optimization Vaccine development 1600 Vicm o Cancer gt 5 Cell biology 40 000 Example Optimization The 96 well electroporation system is ideal for optimizing transfection of eukaryotic cells In this example HELA cells were transfected with a plasmid expressing luciferase The adherent cells were harvested in a log phase of growth and washed twice in Cyto Pulse low conductivity medium The luciferase Luciferase RLU 6 Pulses 3 Pulses 1 Pulse 200 400 expressing plasmid was added to the cells at a cell Pulse Width microseconds density of 1 6 million cells ml and a DNA L concentration of 50ug ul The cell plasmid mixture was added at 200 ulwell to all wells of three rows of a square well 96 well electroporation plate Pulses of 1600 V cm were delivered at varying pulse widths and numbers of pulses Twelve different pulse combinations with three replicates each were delivered A
3. useful for most electroporations and will insure maximum array life The cleaning procedure is also given in Appendix C Sterile plates are available for one time use IMPORTANT WHEN THE 96W A ELECTRODE ARRAY IS REMOVED FROM THE MICROPLATE CAPILLARY ACTION DRAWS SOME LIQUID BETWEEN THE CLOSELY SPACED ELECTRODES WHICH STRADLE THE WALLS THE ARRAY MUST BE CLEANED AND DRIED PRIOR TO REUSE OR THE LIQUID WILL CAUSE AN ELECTRICAL ARC WHEN PULSED THAT MAY DAMAGE THE ARRAY 3 2 4 Washes and other preparatory needs It is important that all tissue culture medium be washed from the cells prior to electroporation This requires a minimum of one centrifugation plus two washes in Cytofusion Medium It is advisable to do the washes immediately prior to electroporation Electroporation medium is non toxic but cells will become fragile after excessive time gt 1 hour in the medium 3 2 5 Electroporation Pulse Electroporation is done by applying one or more high voltage pulses to the cells inducing permeability in the cell membranes The voltage required must be above a threshold to induce membrane breakdown and below a maximum voltage causing cell death Threshold voltage is approximately 0 5 1 5 volts across the cell membrane 1 The voltage across a cell is approximately equal to V 15 r Ecos volts where r cell radius in cm E electric field intensity in v cm angle of the membrane in relation to the direction of the field Cyt
4. 100 ul lysis buffer with DTT to each well Mix each well by pipetting up and down 12 times Add 75 ul lysed cell suspension to wells of a white assay plate for each sample Standard curve if used Remove luciferase standard from 70 C freezer 1 mg ml Add 400 lysing solution to each of 9 tubes Add 1 ml lysing solution to 1 tube Tube A Add 10 ul luciferase to tube A vortex 1 100 dilution Transfer 10 ul from tube A to the first of the 9 tubes 1 41 dilution vortex Transfer 200 ul to the second of 9 tubes 1 3 dilution Serially transfer 200 ul to the remaining tubes use last 8 tubes discard first two Add 75 ul standard to duplicate wells of the white plate Place 50 ul reagent A in each well Place reagent B in Luminometer injector and prime Inject 50 ul Read for 0 5 seconds per well Demonstration of K562 Cell Transfection Using 100000 80000 60000 Luciferase RLU 40000 20000 96 W System 2000 V cm mH 100 us 200 us T m 400 us L 800 us T 1 Pulse 3 Pulses 6 Pulses Number of Pulses versus Pulse Width Figure 4 1 Example K562 Protocol results 4 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 5 Customer Service 5 1 Limited Warranty CYTO PULSE products are warranted against defect in materials and workmanship If the customer
5. Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 2 PA 4000 PA96W Set Up and Operation 2 1 Introduction The PA 96W is an attachment to the PA4000 PulseAgile electroporator It provides the capability of directing the pulse from the electroporator to a well s in the 96 well microplate The method of addressing each well with one or more pulses is presented in Appendix B The system consists of the following PA 4000 Electroporator PA 96W High voltage pulse switch 96W A Electrode array 96W B Base unit 96W P Microplate LCMC CytoFusion Medium CS OPT Interface Cable Set Figure 2 1 The 96 Well Electroporation System PA 4000 PA 96W 2 2 Connecting the PA 96W to the PA 4000 Follow the instructions in Chapter 4 of the PA 4000 manual to set up the PA 4000 The PA 96W should be placed on top of the PA 4000 Figure 2 2 shows the connections to be made The Mains Line Power Switch must be off and the Main Line cord unplugged from the PA 4000 e Connect the Option Interconnect cable between the DB25 connectors on the back of the PA 96W and the PA 4000 DO NOT ATTACH TO A COMPUTER PARALLEL PORT OR A PRINTER e Connect the high voltage cable between the MHV connectors on the back of the PA 96W and the PA 4000 This cord delivers the pulses generated by the PA 4000 to the PA 96W DO NOT USE A CABLE WITH BNC PLUGS e Connect the large high voltage cable with the multi pin connector from the 96W
6. O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 Table of Contents page 1 Introduction 1 1 2 PA 4000 PA 96W Set up and Operation 2 1 2 1 Introduction 2 1 2 2 Connecting the PA 96W to the PA 4000 2 1 2 3 PA 96W Front panel functions 2 2 2 4 Electrode Array Base and Microplate 2 2 2 4 1 96W A Electrode Array 2 2 2 4 2 96W P Microplate 2 2 2 4 3 96W B Base Unit 2 2 2 44 Operating the Base Unit 2 2 2 5 Turning the System on 2 3 2 6 Software 2 3 3 Getting Started with the PA 96W 3 1 3 1 Introduction 3 1 3 2 Preparation for electroporation 3 1 3 2 1 Polynucleotide concentration and cell density 3 1 3 2 2 Medium 3 2 3 2 3 Cleaning and sterilization of the electrode before use 3 3 3 2 4 Washes and other preparatory needs 3 3 3 2 5 Electroporation Pulse 3 3 3 2 6 Programming pulse protocols 3 4 3 3 Sample protocols 3 3 3 3 1 Protocol Design Rules 3 6 3 4 Results using similar protocols 3 6 4 PA 96W Protocol Programming Example 4 1 4 1 Introduction 4 1 4 2 Components 4 1 4 3 Programming a protocol 4 1 4 3 1 General programming guidelines 4 1 4 3 1 1 Viewing programmed protocol 4 1 4 3 1 2 Changing deleting or adding protocol parameters 4 2 4 4 Sample protocol 4 2 4 4 1 Preparation 4 2 442 Materials List 4 2 4 4 3 Harvest cells 4 2 4 44 Cell processing 4 3 4 4 5 Electroporation 4 3 44 6 Harvesting processed cells 4 3 4 4 7 Culture of cells 4 3 4 4 8 Luciferase assay at 24 48 hours 4 4 5 Customer Ser
7. On and HV Off green LEDs are illuminated on the front of the PA 4000 Nothing will illuminate on the front panel of the PA 96W Software Details of software installation and operation of the PA 4000 are described in Chapter 4 of the PA 4000 manual The software that operates the PA 96W is included in the PA 4000 application When the PA 96W Interface cable is connected the software is available Open the PulseAgile application software If the PA 96W is connected properly the application software will recognize the unit There will be three check marks at the top right of the screen At the right bottom there is a portion of the screen called custom In this area are the Three Units which address each row of the eight rows Unit 1 and columns Units 2 and 3 Open a protocol If the user has developed protocols previously just open the protocol folder and click on the desired protocol If this is the first time then open one of the supplied protocols such as PA96W Test pro PA 4000 Interface Version 2 01 C Program Files PulseAgile protocol 0 ptimization 2000 demo pro File Tools Settings Help TIME 00 00 a Sta Rest Options Connected 1W2M3T4 PPS Pro Pulse GROUP LIST I AC Generator I 3 1 Transformer ELECTROPORATION s PRO PULSE SWITCH stem Custom Ok CommLink Clear List for ens lear Lis OK Electrode Holder 0 125 OFF High Voltage Status Local Remote Remote Monitors Power Supply Replace V
8. PulseAgile software Click Electroporation Pro Pulse Switch The window on the left side of the screen has icons for file saving and opening printing communication status and a calculator Open a saved protocol several are supplied with the application Figure 2 2 shows the screen after opening a protocol The center section contains entry sites for the electrical pulse parameters The number of pulses the pulse voltage the pulse duration and the interval after each pulse are programmed here Parameters must be stored in the group to be delivered during running of the protocol There are three actions that can be taken The parameters can be saved to the selected group by clicking the Replace button The parameters can be added as a new group at the end of the list by clicking the Add button The last group can be removed by clicking on the last group then clicking the Remove button Changes made in this section must be saved before moving to another section or the changes will be lost The section in the lower right of the screen labeled 96 Well PPS Mode is the place where the pulses are addressed to wells This section shows an 8 X 12 array for a 96 well plate The buttons above the array allow selection of one or more columns to address the pulses The buttons to the left of the array allow selection of one or more rows Only columns or rows can be selected at any one time That is selection of columns and rows at the same time is not po
9. amplitudes other than those set may be delivered 2 When changing voltage from higher to lower between groups always allow at least a one second pulse interval This may require splitting each group into two groups if pulse intervals other than 1 second are desired For instance to program a pulse protocol to deliver six pulses with 0 125 seconds between each pulse the following will have to be programmed Five pulses with 0 125 second pulse interval plus one pulse with a one second pulse interval Pulse intervals are actually the time from the beginning of a pulse until the beginning of the next pulse Thus the delay occurs after a pulse 3 4 Results using similar optimization protocols Two developmental optimization protocols similar to the supplied sample protocols were tested on different cell lines The Opt S protocol was tested using K562 cells and the Opt N protocol was tested using HELA cells The exact procedure used is listed in chapter 4 Briefly cells were mixed with a luciferase expression plasmid at a concentration of 50 ug ml The K562 cells were electroporated at 5 million cells ml and the HELA cells were electroporated at 1 6 million cells ml The volume used in each electroporation well was 200 ul The results are presented in Figure 2 2 There are several trends evident in this data For the K562 cells the best protocols were 3 or 6 pulses of 2000 V cm and 200 us duration In general better results were obtained in the 200
10. bottomed wells into which the electrodes are inserted 2 4 3 96W B Base Unit Figure 2 3 The 96W B Base 96W A Array The Base Unit is connected to and 96W P Microplate the PA 96W via a long cable which carries 20 high voltage pulse lines The pulse lines are terminated at gold spring loaded contacts The interlock system does not permit the high voltage to be enabled unless the Electrode Array is completely inserted and covering the pulsed high voltage gold contacts 2 4 4 Operating the Base Unit First the Electrode Array is inserted into the Base Next the microplate is inserted with the contents filled Both the Array and Plate must be totally inserted into the Base Then the two handles must be pulled forward which raises the plate tray in the base At the moment of engagement the force required to pull the handle will increase A steady motion should be maintained If the force is too great stop and make sure both the Plate and Array are fully inserted Finally the lid should be closed 22 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 5 PA 96W User Manual Rev 1 11 04 Turning the System on After the set up described in Chapter 4 of the PA 4000 manual is complete and this chapter is read the unit is ready to be used The following steps need to be completed 2 6 Plug the Main Line cord into the PA 4000 Turn on the Main Line Switch on the front of the PA 4000 Check that the Power
11. provides notice of such a defect during warranty period CYTO PULSE at its option will either repair or replace the products which were found to be defective The limited warranty set forth above is exclusive and no other warranty whether written or oral is expressed or implied CYTO PULSE specifically disclaims implied warranties of merchantability and fitness for a particular purpose EXCEPT AS SET FORTH ABOVE CYTO PULSE MAKES NO WARRANTY WITH RESPECT TO THE PRODUCT AND IN NO EVENT REGARDLESS OF CAUSE SHALL CYTO PULSE BE LIABLE FOR INDIRECT SPECIAL OR CONSEQUENTIAL DAMAGES OR OTHER LOSSES OF ANY KIND ARISING FROM BREACH OR WARRANTY OR OTHER USES OF THIS PRODUCT CYTO PULSE S OBLIGATION TO REPAIR OR TO REPLACE TO THE EXTENT SET FORTH ABOVE CONSTITUTES THE EXCLUSIVE REMEDIES OF THE CUSTOMER FOR ANY BREACH OF WARRANTY This warranty shall not apply to products which after inspection by CYTO PULSE were found to be improperly used or to have been modified in any manner CYTO PULSE recommends that the user not open the product cabinet This limited warranty is valid for one year from the date of shipment 5 2 Customer Service If the user believes that there is a defect in the CYTO 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 warra
12. 0 V cm groups With increasing numbers of pulses and pulse widths a peak was reached beyond which results diminished probably due to cell death For the HELA cells better results were obtained with lower electric fields and lower pulse widths Note that the range of pulse widths tested was lower The HELA cells are larger in diameter than K562 cells and these results reflect that difference In general larger cells require lower electric fields and pulse widths than do smaller cells 3 6 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 5 a x o 7 2 i 3 I Luciferase RLU Luciferase RLU 120000 K562 Cell 96W Optimization Protocol Opt S 1 Pulse 100000 Mmm 100 us EX 200 us Hw 400 us EY 800 us 120000 1200 Vicm 1600 V cm 2000 V cm Electric Field versus Pulse Width K562 Cell 96W Optimization Protocol Opt S 3 Pulses 100000 WH 100 us EJ 200 us E 400 us EU 800 us 120000 1200 V cm 1600 V cm 2000 V cm Electric Field versus Pulse Width K562 Cell 96W Optimization Protocol Opt S 6 Pulses 100000 Mim 100 us EX 200 us Tg 400 us E 800 us 1200 Vicm 1600 V cm 2000 V cm Electric Field versus Pulse Width Luciferase RLU Luciferase RLU Luciferase RLU PA 96W User Manual
13. 10 787 1890 v PA 96W User Manual Rev 1 11 04 vi Blank Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 1 Introduction Electroporation has many uses in the fields of cell biology medicine and microbiology The PA 96W add on to the PA 4000 computer controlled electroporator provides the ability to electroporate cells in selected wells of a 96 well microplate All parameters are computer controlled to give the operator maximum flexibility in performing optimization protocols This manual was designed to help you realize the maximum benefit from using the PA 4000 PA 96W system featuring PulseAgile electroporation It contains information on how to operate the electroporator safety tips and protocol optimization Note The PA 4000 with the PA 96W contains a high voltage power supply and was designed with safety features to protect the user and the equipment If used properly the PA 4000 with the PA 96W is a safe and reliable instrument Chapter 3 explains some important concepts related to operator safety in addition to concepts needed for accurate use of the instrument Chapter 3 must be read before setting up this instrument Our goal is the safe and productive use of the PA 4000 with the PA 96W Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 1 1 PA 96W User Manual Rev 1 11 04 Blank Page 1 2 Cyto Pulse Sciences Inc P O
14. 6 Well Driver User Manual Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 1 410 787 1890 1 410 787 1891 FAX www cytopulse com Cyto Pulse Sciences Inc makes no warranty with respect to the product except for the warranty set forth in this Users Manual on page 5 1 The LIMITED WARRANTY SET FORTH ON PAGE 5 1 IS EXCLUSIVE AND NO OTHER WARRANTY WHETHER WRITTEN OR ORAL IS EXPRESSED OR IMPLIED Cyto Pulse Sciences Inc SPECIFICALLY DISCLAIMS IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE License Agreement PA 96W Programmable 96 Well Driver Cyto Pulse Sciences Inc Licensor conveys to the licensee for fee paid a nonexclusive nontransferable license to use the PulseAgile hardware and software equipment for research purposes into perpetuity Cyto Pulse Sciences Inc has patent allowance and patents pending covering the PulseAgile and other process If the licensee wishes to use the hardware and software for production or commercial purposes an additional license shall be required The equipment is not approved by the FDA foruse with in vitro or in vivo diagnostics or therapy The information in this manual is subject to change without notice Copyright 2000 2005 Cyto Pulse Sciences Inc PAS6WUMANrev 1 1 1 04 Price 100 00 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 i PA 96W User Manual Rev 1 11 04 Blank Page ii Cyto Pulse Sciences Inc P
15. B Base to the PA 96W e Connect the small cable with the RCA phono style connector from the 96W B Base to the Interlock jack on the back of the PA 4000 Figure 2 2 Back Panel Connections Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 2 1 PA 96W User Manual Rev 1 11 04 2 3 PA 96W Front Panel Functions There are 96 indicators on the front panel Each indicator will illuminate red or green depending on the direction of electric field in the well One of the unique features of this system is the electric field in the well can be reversed from one pulse to the next If both electrodes in the well are the same plus or ground then there is no illumination This unit is powered from the PA 4000 Mains Line Power switch 2 4 Electrode Array Base and Microplate The three components form a method by which the 192 separate gold electrodes are inserted into the microplate A significant force is required to insure proper insertion That force is supplied by the base unit which applies the force in a very uniform manner across the array 2 4 1 96W A Electrode Array The array consists of 96 pairs of gold plated electrodes mounted in an insulating base There are 12 column lines and 8 row lines and two interlock lines which are terminated in gold plated contacts 2 4 2 96W P Microplate The 96 well microplate is made of injection molded polypropylene and cannot be autoclaved It contains 96 square flat
16. Rev 1 11 04 HELA Cell 96W Optimization Protocol Opt N 1 Pulse E 50 us EY 100 us E 200 us II 400 us 1200 V cm 1600 V cm 2000 V cm Electric Field versus Pulse Width HELA Cell 96 W Optimization Protocol Opnt N 3 Pulses HEN 50 us EY 100 us HM 200 us E 400 us 1200 V cm 1600 V cm 2000 V cm Electric Field Versus Pulse Width HELA Cell 96W Optimization Protocol Opt N 6 Pulses EE 50 us EX 100 us HA 200 us EY 400 us I T T 1200 V cm 1600 V cm 2000V cm Electric field versus Pulse Width Figure 3 2 Results of Optimization Protocols on K562 and HELA Cells Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 Blank Page 3 8 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 4 PA 96W Protocol Programming Example 4 1 Introduction The PA 96W system can be used to optimize transfection of selected cell lines or can be used to deliver identical electrical protocols to wells of a 96 well microplate A sample protocol will be presented showing an optimization protocol for K562 cells 4 2 Components Cyto Pulse Sciences Inc PA 4000 with PA 96W accessory Equip
17. TM Cyto Pulse Sciences Inc Model PA 96W Programmable 96 Well Driver User Manual Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 1 410 787 1890 1 410 787 1891 FAX www cytopulse com Cyto Pulse Sciences Inc makes no warranty with respect to the product except for the warranty set forth in this Users Manual on page 5 1 The LIMITED WARRANTY SET FORTH ON PAGE 5 1 IS EXCLUSIVE AND NO OTHER WARRANTY WHETHER WRITTEN OR ORAL IS EXPRESSED OR IMPLIED Cyto Pulse Sciences Inc SPECIFICALLY DISCLAIMS IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE License Agreement PA 96W Programmable 96 Well Driver Cyto Pulse Sciences Inc Licensor conveys to the licensee for fee paid a nonexclusive nontransferable license to use the PulseAgile hardware and software equipment for research purposes into perpetuity Cyto Pulse Sciences Inc has patent allowance and patents pending covering the PulseAgile and other process If the licensee wishes to use the hardware and software for production or commercial purposes an additional license shall be required The equipment is not approved by the FDA for usewith in vitro or in vivo diagnostics or therapy The information in this manual is subject to change without notice Copyright 2000 2005 Cyto Pulse Sciences Inc PAS6WUMANrev 1 1 1 04 Price 100 00 PA 96W User Manual Rev 1 11 04 TM Cyto Pulse Sciences Inc Model PA 96W Programmable 9
18. ase Pipette material into microplate through Array no cross over Close safety top of Base and pulse Page 2 A 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 1890 PA 96W User Manual Rev 1 11 04 Appendix B PA 96W Switch and 96W A Electrode Array Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 B 1 PA 96W User Manual Rev 1 11 04 Blank Page B 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 9U seouaIDS asind OAD B 3 HUN L HUN a 8 109 PO H mou PO e O ea O 1109 O 5 Moy HO Q 3 e O 9109 PO0 i 3 moy PO i O O O jo gun SB are zi 109 0 p 109 PbO D a moy PO i Q O gt 6 spun YO MS 11 199 PO 129 O 3 moy PO y O O e O Q O OL 109 Q I z 109 O g moy o M96 Vd O A O O 6109 Q 109 Q Yy Moy ou apIsul l AAA AA suwnjog Aey 0 smoy Aey 0 M96 Wd 0 M96 Vd Wol4 M96 Vd Wo4 000P Vd Wo4 UIJIMS 2JEIS 39141 M96 Vd Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 LELLULLM LED LULLLLIMI A E CULCCULW ELEULUUMI 000t Yd Keli p01 9913 V M96 Q lt o TU D D n o D o los oD i 2 B 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual
19. con e Select Electroporation Pro Pulse Switch e Open the protocol called K562 Demo Opt Click on the open folder icon upper left icon in the set of icons on the left side of the screen Click on K562 Demo Opt Copyright Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 4 1 PA 96W User Manual Rev 1 11 04 4 3 1 2 Changing deleting or adding protocol parameters e Parameters within a group are changed by changing the values on the screen then clicking Replace The Replace button should be clicked after changing pulse parameters For practice click on Group 1 in the Group List Change the number of pulses to 2 by either selecting the increase button under the pulse number or by selecting the number with a mouse and typing the number 2 Click Replace to change the parameters in group 1 Changes will not be permanently saved until the file is saved These changes are not permanently needed so do not save the file Protocol files when needed can be saved by clicking the disk icon and giving the new protocol a name e Any group is deleted by clicking on it then selecting Remove For practice click on group 12 and click Remove e Groups are added by clicking Add This copies all parameters shown on the screen into a new group that will be added at the end of the group list Parameters in that group can then be changed by changing then values on the screen and clicking replace For practice click on A
20. dd to add a new group Change the number of pulses in this group to 8 and click Replace 4 4 Sample protocol 4 4 1 Preparation e Expand K562 Cells to two T 150 flasks log growth medium to high density e Obtain all items in the Materials List 4 4 2 Materials List e Cyto Fusion Medium e Plasmid Expressing Luciferase e Complete Medium DMEM FBS 10 L glutamine Penicillin Streptomycin 96W P 96 well electroporation plate 96 well culture plate clear tissue culture flat bottom 96 well assay plate white opaque 50 ml conical tubes Luciferase reagent Luciferase Assay Kit Tropix Inc BC300L Reagent A Reagent B Lysis buffer Make sure that DTT has been added to the lysis buffer 4 4 3 Harvest cells e Select two T 150 flasks of K562 Cells e Place cells in 50 ml conical tubes 4 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 4 44 Cell Processing Centrifuge cells 1500 rom X 10 minutes Add 20 ml total Cytofusion Medium Centrifuge cells 1500 rom X 10 minutes Add 20 ml total Cytofusion Medium Centrifuge cells 1500 rom X 10 minutes Re suspend in 7 ml Cytofusion Medium Count X Dilution X 10 000 X Volume Total Cells Re suspend cells in Cytofusion Medium at a concentration of 5 million cells per ml Total cells 5 000 000 cells ml ml Calculate amount of cells needed _ wells X 0 2 ml well _ ml 86 million cells Add plasmid DNA to a fi
21. e Soak array for 10 minutes in 70 Isopropanol Air dry in laminar flow hood Sterilization Array may be gas sterilized using ethylene oxide gas DO NOT AUTOCLAVE OR EXPOSE TO DRY HEAT C 3 PA 96W User Manual Rev 1 11 04 Blank Page C 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890
22. e of these principles will be reviewed in this chapter 3 2 Preparation for electroporation Prior to beginning a transfection using the 96 Well system several factors need to be considered The condition of the cells is the most important Cells should have been recently passed and in a log phase of growth For suspension cells this means that a less than maximum cell density must be used For adherent cells less than 90 confluency is recommended In addition cells will change in their response to electroporation based transfection with increasing passage number Some cell lines become more resistant to transfection with increasing cell line passage Although this is different for different cells lines it is recommended that the cells used be at a passage level less than 30 Since the equivalent of 96 cuvettes is used with a full 96 well electroporation supplies will be needed for that many transfections For instance if a plasmid DNA concentration of 50 ug ml is used and a cell density of 1 million cells ml is used then 1 mg of plasmid DNA and 20 million cells will be needed using the minimum volume of 200 ul well 3 2 1 Polynucleotide concentration and cell density The underlying principle for considering polynucleotide concentration is that the molarity of the material is important For instance if a plasmid is used at a concentration of 50 ug ml with a cell density of 1 million cells ml then the same plasmid will be used at a concen
23. fter culture for 24 hours luciferase production was assayed A pulse width of 100 us with either three or six pulses gave optimal results The electroporations and analyses were done by Alan D King D V M Ph D The photomicrograph shows HELA cells transfected using a DsRed Clontech reporter plasmid The pulse protocol was 6 pulses of 1600 V cm 400 us pulse width and 0 125 second e y interval between pulses A high percent of the cells show e mail aston expression of DsRed Photograph courtesy of Shigeyoshi Fujiwara MD PhD National Research Institute for Child Health Feb 04 and Development Tokyo Japan Page Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 A 3 PA 96W User Manual Rev 1 11 04 Cyto Pulse Electroporation and Electrofusion Systems for In vivo therapeutic delivery Gene therapy Immunotherapy Hybridoma production Vaccine delivery Nuclear transfer mRNA delivery siRNA delivery plasmid delivery Headquarters P O Box 609 Columbia MD 21045 USA Voice 410 715 0990 Fax 410 715 2148 Web www cytopulse com e mail customer cytopulse com Jan 04 Cyto Pulse Sciences Inc A medical device and treatment development company Data Sheet 96 Well Electroporation System for Eukaryotic Cells The Cyto Pulse 96 Well Electroporation System Includes e The PA 40005 Advanced Electroporation System which produces both standard rectangular square pulses and P
24. ing these guidelines 10 20 million cells ml is a safe cell density Cells densities up to 40 million cells ml have been used Cells in Cyto Pulse s low conductivity medium must be diluted at least 1 5 in complete cell culture medium after electroporation Adherent cells can be cultured for short periods with as little as a 1 2 dilution followed by complete removal and replacement after adherence to the tissue culture vessel The requirement for this dilution may limit the practical minimum density of cells 3 22 Medium Cells need to be in a low conductivity medium for two reasons One reason is that the 96W system can pulse more that one well at a time This means that there is more electrical current required from the pulse generator when more wells are pulsed simultaneously There are limits on the maximum current that can be delivered and the use of low conductivity medium keeps the current demand below maximum limits Second the high current generated in a highly conductive media will cause excessive heating The temperature in high conductivity medium can rise to critical levels and kill the cells Conductivity is used to describe the resistance of the medium because conductivity takes into account the volume of liquid in a well or cuvette Remember that for a parallel plate electrode conductivity is related to resistance ohms by the formula 2 R 2 lg ohms o Ao vol where o Conductivity in S cm 1 p I plate spacing A
25. luded with the software are K562 Demo pro and the Opt N series The Opt N series is a set of three protocols that can be used for initial optimization The Opt N series forms a grid of electric field 2000 v cm 1600 v cm and 1200 v cm Pulse Width 50us 100us 200us and 400us and numbers of pulses 1 3 and 6 This 3X4X3 matrix is divided among 3 protocols based upon the number of pulses They are called Opt N 1 Pulse Opt N 3 Pulse and Opt N 6 Pulse When one of these protocols is run either the field intensity or pulse width is changed in each column of the plate All eight rows of each column are identical providing an 8 replicate sample Sample protocol Opt N 1 Pulse pro all 8 rows in a column pulsed simultaneously Col Group PA PW Pl PN volts us sec 1 1 1080 50 125 1 2 2 1080 100 125 1 3 3 1080 200 125 1 4 4 1080 400 125 1 5 5 865 50 125 1 6 6 865 100 125 1 7 7 865 200 125 1 8 8 865 400 125 1 9 9 650 50 125 1 10 10 650 100 125 1 11 11 650 200 125 1 12 12 650 400 125 1 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 3 3 1 Protocol Design Rules 1 If a Protocol is designed that changes voltage from one group to the next it should always be setup so the voltage change is decreasing between the groups If the programmed voltage is increasing between groups pulse
26. ment must be set up according to instructions given in Chapter 2 4 3 Programming a protocol 4 3 1 General programming guidelines Start the program by clicking on the PulseAgile Icon Select Electroporation Pro Pulse Switch in the upper left of the screen The fastest way to get started programming is to open a pre programmed protocol and modify desired parameters For each group the following parameters are programmed Pulse voltage Pulse width Number of pulses in a group Pulse Interval timed after each pulse including the last one in a group Wells to apply pulse Direction of electric field in wells in Custom mode only When all pulses in a group have been delivered the software selects the next group in the list and executes those pulses Some rules must be observed in order for the pulses to be correctly delivered e Plan any changes of voltage so that the voltage is lowered or remains the same as you progress from group to group That is program higher voltage pulses to be delivered first e Entire rows or columns must be selected Any number of columns or rows can be selected from 1 to 8 rows or 12 columns The 96 well graphic in the lower right of the screen makes this selection easy e When decreasing voltage from one group to the next program one second Pulse Interval or follow instructions given in section 3 3 1 paragraph 2 4 3 1 1 Viewing programmed protocol e Start the program by clicking the program i
27. nal concentration of 50 ug ml to the cell suspension 50 ug ml X ml ug DNA Divide by ug ul ul Mix well and dispense 200 ul cell suspension to each well in three rows of the square well 96 well electroporation plate 4 4 5 Electroporation Set up the PA 4000 electroporator PA 96W and base according to manual See Chapter 2 Program the following design all at 2000 V cm 0 125 S pulse interval Column1 1080 V 100us 1 Pulse Column2 1080 V 200 us 1 Pulse Column 3 1080 V 400 us 1 Pulse Column 4 1080 V 800 us 1 Pulse Column 5 1080 V 100us 3 Pulses Column6 1080 V 200 us 3 Pulses Column 7 1080 V 400 us 3 Pulses Column 8 1080 V 800 us 3 Pulses Column 9 1080 V 100us 6 Pulses Column 101080 V 200 us 6 Pulses Column 111080 V 400 us 6 Pulses Column 121080 V 800 us 6 Pulses Note This design is called K562 Demo pro in your protocol list Simply open that file to input these parameters Other optimization protocols are described in the Chapter 3 4 4 6 Harvesting processed cells Transfer all cells from electroporation plate to a second 96 well plate Mix well and transfer 30 ul cells to identical wells of a 96 well assay plate Add 120 ul Complete Medium well 4 4 7 Culture of cells Incubate at 37 C for one to two days 5 CO Copyright Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 4 3 PA 96W User Manual Rev 1 11 04 4 4 8 Luciferase assay at 24 48 hours 120000 Add
28. nty period is still in effect the user will be given an authorization number RMA to return the product If after receipt and inspection the product is found to be defective it will be replaced or repaired and returned to the customer If the product is found to have been modified or misused the user will be given a quote for repair If the warranty period has expired and the user requests repair CYTO PULSE will inspect the product and provide a written quote for repair The user must provide a purchase order number before the product will be repaired If the unit is damaged in shipment the user must recover the insured value to replace or repair from the carrier Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 5 1 PA 96W User Manual Rev 1 11 04 Blank page 5 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 Appendix A Data Sheet Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 A 1 PA 96W User Manual Rev 1 11 04 Blank Page A 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 715 1890 PA 96W User Manual Rev 1 11 04 Cyto Pulse Sciences Inc A medical device and treatment development company D ata Sh e et 96 Well Electroporation System for Eukaryotic Cells Fast Rectangular Wave Electroporation Optimization e Pulse Parameters amplitude width number interval Cell density
29. o Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 3 3 PA 96W User Manual Rev 1 11 04 Electric fields 2 3 times threshold can be used This should be optimized for the cells used Multiple electroporation pulses may be more efficient than a single pulse A special pulse waveform called PulseAgile is available This option is only available on the Cyto Pulse PA 4000 Electroporator including the PA 96W system PulseAgile means the ability to change pulse parameters from pulse to pulse in the same well or cuvette It is normally done by delivering one or more high electric field pulses followed by two or more lower electric field pulses The higher electric field pulses induce permeability in a large area of the cell surface The lower electric field pulses help move charged polynucleotides into cells by electrophoresis 3 2 6 Programming pulse protocols Programming the pulse protocols for the PA 4000 PA 96W system consists of programming the electrical pulse parameters and the address where the system should deliver those pulses These parameters are stored in a stack called a Group List Each group in the list describes one set of electrical pulse parameters plus one well address The groups are shown in a window on the software screen As each group is completed the next group is begun Figure 2 1 on the next page shows an example screen Begin with proper set up of the equipment and turn all equipment on Open the
30. oltmeter 10 volts Load Remove Ohmmeter ohms Last Protocol Log 96 Well PPS Mode All EE oon ae sel UA 2 U m m eee I nImOnNner ss u Figure 2 4 Set up Screen PA 96W with Protocol Opened Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 23 PA 96W User Manual Rev 1 11 04 Blank Page 9 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 3 Getting Started with the PA 96W 3 1 Introduction Electroporation using the PA 4000 PA 96W system increases the capacity to do a large number of electroporations Each 96 well plate is equivalent to 96 individual cuvettes This chapter introduces concepts behind 96 Well electroporation and provides information to allow optimal use of the system The system consists of the PA 4000 Electroporator PA 96W Switch 96W A Electrode Array 96W B Base and 96W P Microplate Several example experimental designs that can be used with 96 Well electroporation are e Optimization using up to 12 different pulse waveform protocols e Electroporation of different cell types in the same 96 well plate each requiring different pulse protocols e Electroporation using different plasmids mRNA siRNA or other material in each well There are of course many other experimental designs possible The principles of electroporation are covered in detail in the accompanying PA 4000 manual Som
31. plate area vol volume of liquid A Cyto Pulse Cytofusion Low Conductivity Medium C CPS LCMC has a conductivity of 80 uS cm Conductivity is the reciprocal of resistivity which is 12 500 Q cm To obtain a medium with a high resistivity or low conductivity a low ionic medium formula is used 3 2 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 Another important property of the electrofusion medium is the osmolarity The osmolarity of electroporation media is usually iso osmolar normal osmolarity of body fluids 290 mOs Cyto Pulse Cytofusion Medium C is an optimized iso osmolar electrofusion medium designed for optimal results Cyto Pulse recommends that this medium be used for electroporation 3 2 3 Cleaning and sterilization of the electrode before use The 96W A Electrode Array and the 96W P square well plate are not autoclaveable The recommended cleaning procedure is Rinse the array immediately after use with high quality water e Ifa film or material are present on the electrode wash with a mild soap solution or Windex A soft toothbrush can be used e Rinse thoroughly after cleaning with high quality water e For sterilization Soak for 10 minutes in 3 hydrogen peroxide or Sporeklenz Rinse in 70 alcohol then soak for 10 minutes in 70 alcohol Air dry in a laminar flow hood The latter procedure will provide a well sanitized or sterilized array
32. ssible Once the pulse parameters have been selected including row or column addressing one more action must be taken The parameters must be either added to a new group or replace values in an existing group The parameters are added to a new group by selecting the Add button at the top of the group list The parameters replace the values in a group by selecting Replace at the bottom of the group list A detailed protocol described in Chapter 4 will provide some practice in modifying parameters 3 4 Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 PA 4000 Interface Version 2 01 File Tools Settings Help TIME 00 00 96 Well Custom Clear List ELECTROPORATION PRO PULSE SWITCH Last Protocol Log E I IMmMONUr CACA ee u Figure 3 1 3 3 Sample protocols C Program Files PulseAgile protocol O ptimization 2000 demo pro Rest Options Connected INTERVAL Pro Pulse GROUP LIST M1hM263 7 4 PPS I AC Generator FF 3 1 Transformer System Ok CommLink Ok Electrode Holder fo FF High Voltage Ready Status Local Remote Remote Monitors Power Supply E 10 volts ohms Replace Voltmeter Load Ohmmeter 96 Well PPS Mode 12345670 91001 T m m m m n a n a a a a PulseAgile Screen After Opening a 12 Group 96W Protocol Several protocols are supplied pre programmed for your use or modification Inc
33. tration of 50 ug ml if the cell density is changed to 10 million cells ml Typical concentrations used are e Plasmid DNA 25 50 ug ml e siRNA 1 to 100 nM e mRNA 1 to 20 pg ml These polynucleotide concentrations are just examples Concentrations outside these ranges are widely reported in the literature Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 3 1 PA 96W User Manual Rev 1 11 04 There are several factors to consider when deciding on the cell density Cell density in culture prior to electroporation is dependent upon achieving a log phase harvest This is a critical factor in achieving successful polynucleotide delivery Cell density for culture after electroporation will depend upon the needs of individual cells Adherent cells for instance will need space on the plastic to adhere and to multiply Cell density during electroporation is more flexible The lower limit of cell density is dependent upon having enough cells to assay and culture after electroporation Generally we use at least 0 5 million cells per ml However there is no theoretical reason why a lower cell density could not be used The upper limit of cell density depends upon 1 The percent volume occupied by the cells lt 1 recommended 2 the amount of ions leaking from cells affects medium conductivity 3 The amount of cells available 4 Whether or not the low conductivity medium is left in culture after electroporation Us
34. ulseAgile Pulse waveforms o User interface Windows based software o Users Manual o Cuvette Holder e The PA 96W Pulse Switch Option to the PA 4000 Electroporator o PA 96W Programmable Pulse Switch controlled by the system software interface o 96W A Reusable electrode Array with 96 pairs of gold plated electrodes o 96W B Base Unit which holds the microplate and electrode array o 96W P 96 well polypropylene microplate 5 each non sterile Two meter cable to place Base Array Microplate in safety hood e One 500 ml bottle low conductivity medium The laptop is not included The PA 4000 Electroporator has the CE Mark Compliance approval for the PA 96W Programmable Switch is expected in 2004 CE CB FCC 96W A The 96 Electrode Pair Array The array has 96 pairs of electrodes which are designed to fit into the special square well flat bottom microplate The performance specifications are Volume Volume recovered Volume recovered Volume E Field GF Array Inserted Array Removed Treated Variation pl cm 200 gt 80 gt 95 gt 95 lt 5 1 480 400 gt 85 gt 95 gt 95 lt 5 0 740 Columns with rows in columns averaged includes pipetting error GF is the geometric factor of the parallel plates which includes gap and area gap _ GF ohms G conductivity in S cm vol volume in ml gap 0 54 cm o vol o well T Operating Procedure Insert microplate in Base Insert Electrode Array in b
35. vice 5 1 5 1 Limited Warranty 5 1 5 2 Customer Service 5 1 Appendix A Data Sheet Appendix B PA 96W Switch and Electrode Array Connections Appendix C Electrode Array Cleaning Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 iii PA 96W User Manual Rev 1 11 04 List of Figures The 96 Well Electroporation System Back panel Connections The 96W B Base 96W A Array and 96W P Microplate Set up Screen PA 96W with Protocol Open PulseAgile ScreenAfter Opening a 12 Group Protocol Results of Optimization Protocols on K562 and HELA Cells Example K562 Protocol results Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 410 787 1890 PA 96W User Manual Rev 1 11 04 Caution Notice This instrument contains a high voltage power supply adjustable beyond 1 000 volts such voltage can be lethal The user must read this manual carefully before the instrument is placed into operation Removing the cover may void the warranty Do not connect or disconnect the high voltage cable with the high voltage enabled To connect or disconnect the cable turn line mains power off and unplug the line mains cord Do not open the cuvette holder while the high voltage is on If a problem occurs during a run push the STOP RESET button on the front panel If there are any questions about the operation of this instrument call Cyto Pulse Customer service Cyto Pulse Sciences Inc P O Box 609 Columbia MD 21045 USA 4

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