BTX AgilePulse ID User manual

AgilePulse™ID
In Vivo
Gene Delivery System
USER’S MANUAL
Publicaon 015-101444 Rev 2.0

Thank you for invesng in an AgilePulse system.
Since its founding in 1983, the main focus of BTX has been in the area of applying
controlled electric elds for genec engineering applicaons. Because of this,
we quickly established a reputaon as the technological leader in the elds of
electroporaon and electrofusion. Our systems have been installed in many
presgious instutes around the globe where they are used successfully for high
eciency transfecon, transformaon and cell fusion applicaons. We oer a
variety of waveforms, electrodes and chamber opons to provide you with the
best tools to achieve your goals.
We are vested in your success. To that end, the BTX technical support team
constantly tracks published literature for any reference to electroporaon and
electrofusion. We extract the pernent experimental condions and yields
from these papers to help us in our eorts to help you. In addion to tracking
publicaons, we are available to you for support at any me for advice in
experimental design, product recommendaons, troubleshoong, and any other
relevant technical advice.
We thank you again for your investment and we look forward to assisng you in
any way we can.
Finally, please read this manual carefully before aempng to operate the
electroporaon system. If you have any quesons about the unit or about
parcular applicaons, please contact us:
BTX
84 October Hill Road
Holliston, MA 01746 USA
Toll Free: 1-800-272-2775
Internaonal Callers: 508-893-8999
Fax: [phone]
Web: www.btxonline.com
Email: [email protected]
For any customers outside the US or Canada, please call your local BTX dealer or
call us directly.
A Message from BTX
AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com

1
AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Table of Contents
SUBJECT PAGE #
Warranty Informaon 3
Safety Informaon 4
AgilePulse™ ID System Components and Set-Up 5–7
Introducon 5
The AgilePulse Waveform Generator 5
Front Panel 6
Back Panel 6
Intradermal Electrode Array (ID) 6
Seng up the System 7
Inial System Test 7
System Operaon 8–16
Experiment Delivery 8
USB Key/Memory Sck 8
Intradermal Array (IDA) Electrode 8
Handle 8
Electrode Tip 8
Waveform Generator 9
Login Screen — User Idencaon Input 9
Welcome Screen — Idencaon Input 9
Experiment Delivery Screen Funcons and Operaon 10
Experiment Delivery Logs 11
Text Log 12
CSV Data 13
Delivery Compleon Return Codes 14
Soware Maintenance Mode 15
User Account Maintenance 15
Log Retrieval 16
Pulse Protocol 16
AgilePulse™ ID Tutorial 17–26
Applicaon 17
Experimental Delivery Methods 17
Delivery into Cells Using Electroporaon 18
General Electroporaon Discussion 18
AgilePulse™ Electroporaon 20–22
AgilePulse In Vivo/Ex Plant Electroporaon Protocol
(Animal Skin/Tibialis Muscle)
23–24
Specicaons 25
Maintenance 26
Fuse Replacement 26
Electrode Care and Cleaning 26
Ordering Informaon 27
References 28–29

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Table of Contents
LIST OF FIGURES PAGE #
Figure 1: AgilePulse Waveform Generator 5
Figure 2: AgilePulse Waveform Generator Selecon Guide 5
Figure 3: AgilePulse Front Panel 6
Figure 4: Login Screen 7
Figure 5: System OK Screen 7
Figure 6: Self Check Not Successful Screen 7
Figure 7: IDA Electrode Handle 8
Figure 8: IDA Tip 8
Figure 9: Aaching IDA Tip to Handle 8
Figure 10: User Login Screen 9
Figure 11: Opening Screen 9
Figure 12: Delivery Screen — Inializaon 10
Figure 13: Delivery Screen — Load Esmaon 10
Figure 14: Delivery Screen — High Voltage Power Supply Charge 10
Figure 15: Delivery Screen — Pulse Waveform Acve 10
Figure 16: Delivery Screen — Shutdown 11
Figure 17: Text Log Screen 12
Figure 18: Sample Amplitude Analysis 13
Figure 19: User Account Screen 15
Figure 20: Log Retrieval Screen 16
Figure 21: Pulse Protocol Modicaon 16
Figure 22: Electroporaon 18
Figure 23: Transmembrane Voltage 19
Figure 24: Two Needles per Row Field 22
Figure 25: Electrical Field Coverage 23
Figure 26: Bleb Formaon 23
Figure 27: Fuse Replacement 26

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Warranty Information
RESEARCH ONLY
BTX
84 October Hill Rd
Holliston, MA 01746, USA
Phone: [phone]
Fax: [phone]
Web: www.btxonline.com
Warranty
BTX warranes AgilePulse systems for a period of two years from
the date of purchase. At its opon, BTX will repair or replace
the product if it is found to be defecve as to workmanship or
materials. This warranty does not extend to any device which
has been (a) subjected to misuse, neglect, accident or abuse, (b)
repaired or altered by anyone other than BTX without BTX express
and prior approval, (c) used in violaon of instrucons furnished
by BTX. This warranty extends only to the original customer
purchaser. IN NO EVENT SHALL BTX BE LIABLE FOR INCIDENTAL OR
CONSEQUENTIAL DAMAGES. Some states do not allow exclusion
or limitaon of incidental or consequenal damages so the above
limitaon or exclusion may not apply to you. THERE ARE NO
IMPLIED WARRANTIES OF MERCHANTABILITY, OR FITNESS FOR
A PARTICULAR USE, OR OF ANY OTHER NATURE. Some states
do not allow this limitaon on an implied warranty, so the above
limitaon may not apply to you. Without liming the generality
of the foregoing, BTX shall not be liable for any claims of any kind
whatsoever, as to the equipment delivered or for non-delivery of
equipment, and whether or not based on negligence. Warranty
is void if the BTX AgilePulse system is changed in any way from
its original factory design or if repairs are aempted without
wrien authorizaon by BTX. Warranty is void if parts, connecons
or electrodes not manufactured by BTX are used with the BTX
AgilePulse instrument. If a defect arises within the warranty period,
promptly contact BTX, 84 October Hill Road, Building 7, Holliston,
Massachuses, USA 01746-1388 using our toll free number
1-800-272-2775 (US Only) or 508-893-8999 (E-mail: support@
hbiosci.com). Goods will not be accepted for return unless an RMA
(Returned Materials Authorizaon) number has been issued by
our customer service department. The customer is responsible
for shipping charges. Please allow a reasonable period of me
for compleon of repairs, replacement and return. If the unit is
replaced, the replacement unit is covered only for the remainder
of the original warranty period dang from the purchase of the
original device. This warranty gives you specic rights, and you may
also have other rights, which vary from state to state.
Out of Warranty Service
Proceed exactly as for Warranty Service above. If our service
department can assist you by phone or other correspondence, we
will be glad to help at no charge.
Repair service will be billed on the basis of labor and materials.
A complete statement of me spent and materials used will be
supplied. Shipment to BTX should be prepaid. Your bill will include
return shipment freight charges.
Disassembly by the user is prohibited. Service should only be
carried out by experienced BTX technicians.
Repair Facilies and Parts
BTX stocks replacement and repair parts. When ordering, please
describe parts as completely as possible, preferably using our part
numbers. If praccal, enclose a sample photo or drawing.
Cauon Noce
The BTX AgilePulse systems are intended for laboratory use only
and can be used in research and development applicaons.
These systems have been designed to meet the standards for
electromagnec compability (EMC) and safety intended for
laboratory equipment applicaons.
This product should not be used in the presence of a ammable
atmosphere such as an anesthec mixture with air, oxygen, or
nitrous oxide.

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Caution Risk
of Electric Shock
Caution Protective
Ground Terminal
High Voltage Risk
These instruments contain a high voltage power supply adjustable
to 1,000 V. High voltage power supplies present a serious risk of
personal injury if not used in accordance with design and/or use
specicaons, if used in applicaons on products for which they
are not intended or designed, or if they are used by untrained or
unqualied personnel.
• The user must read this manual carefully before the
instruments are placed into operaon.
• Removing the cover will 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 o and
unplug line (mains) cord.
• Do not touch the electrode p while the waveforms are
being applied.
• Do not under any circumstances turn the power switch o
while the system is pulsing; if an emergency occurs push
the emergency buon on the front panel
If there are any quesons about the operaon of this instrument,
call BTX Customer service at 1-800-272-2775, or 1-508-893-8999.
Cauon Noce
The BTX AgilePulse Systems are intended for laboratory use
only and can be used in research and development applicaons.
These systems have been designed to meet the standards for
electromagnec compability (EMC) intended for laboratory
equipment applicaons as well as the applicable safety
requirements for electrical equipment for measurement, control,
and laboratory use. The unit itself does not generate waste, but
may be used to treat samples that are hazardous. Please use
appropriate PPE and ensure disposal in accordance with local
regulaons and pracces.
This product should not be used in the presence of a ammable
atmosphere such as an anesthec mixture with air, oxygen, or
nitrous oxide.
Please read the following safety precauons to ensure proper
use of your generator. If the equipment is used in a manner not
specied, the protecon provided by the equipment may be
impaired.
To Prevent Hazard or Injury
Emergency Stop
If a problem occurs during a run, push the STOP/RESET buon on
the front panel.
Use Proper Line Cord
Use only the specied line cord for this product and make sure line
cord is cered for country of use. The operang voltage range for
the AgilePulse Series is 100 to 240 VAC, 50/60 Hz.
Ground the Product
This product is grounded through the grounding conductor of the
power cord. To avoid electric shock, the grounding conductor must
be connected to earth ground. Before making any connecons
to the input or output terminals of the product, ensure that the
product is properly grounded.
Make Proper Connecons
Make sure all connecons are made properly and securely. Any
signal wire connecons to the unit must be no longer than three
meters.
Observe All Terminal Rangs
Review the operang manual to learn the rangs on all
connecons.
Use Proper Fuse
Use only specied fuses with product.
Avoid Exposed Circuitry
Do not touch any electronic circuitry inside of the product.
Do Not Operate with Suspected Failures
If damage is suspected on or to the product do not operate the
product. Contact qualied service personnel to perform inspecon.
Orient the Equipment Properly
Do not orient the equipment so that it is dicult to operate the
disconnecon device.
Place Product in Proper Environment
Review the operang manual for guidelines for proper operang
environments.
Observe All Warning Labels on Product
Read all labels on product to ensure proper usage.
Safety Information

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Introducon
The AgilePulse™ system provides the capability of delivering DNA
vaccines to the dermis. The system is designed for easy set-up and
operaon.
Specicaons are presented on page 27.
The AgilePulse™ ID system is composed of three subsystems:
• Touchscreen controlled AgilePulse Waveform Generator
• Intradermal Array (ID) parallel, needle electrodes
• User Manual
Oponal equipment available includes:
• Foot Pedal (47-0420)
• Adapter box for electrodes with 4mm banana cables
(47-0090 or 47-0091, please see Ordering Informaon
page 27 for details)
• 2-Needle Array, 5 mm gap, pkg. of 6 (45-0121)
• 2-Needle Array Handle, for 5 mm gap (45-0206)
The AgilePulse ID
Waveform Generator
The Waveform Generator (Figure 1) is composed of a computer and
high voltage pulsing circuits. The computer runs mobile Windows®
and is operated by a touch screen. On the front panel there is
a power switch, a touch screen, an emergency stop switch, an
electrode connector and two USB ports. All User inputs are entered
via the touch screen or by connecng a keyboard to one of the USB
ports. The system may be operated by using an oponal foot pedal.
On the back panel there is a line/mains power connector.
The high voltage pulsing circuits are controlled by a microcontroller.
The microcontroller accepts user inputs from the touch screen and
produces the pulse waveforms. A second microcontroller is used
as an independent audit of the waveforms produced. If the audit
funcon detects a deviaon from the desired pulse protocol, the
system immediately terminates pulsing. The system also digizes
the output of the pulse voltage and pulse current monitors and
calculates the skin resistance of the ssue on each pulse. This is
a signicant quality control funcon that indicates the vaccine is
being delivered properly. Aer the vaccine is delivered, a log of all
parameters is saved in Internal Memory and on a USB memory key,
if one is inserted.
Figure 1: AgilePulse Waveform Generator
Figure 2
AgilePulse Waveform Generators come in two variees,
specialized for pulse accuracy in relaon to electrical resistance
of the sample ssue. AgilePulse ID is recommended for
electroporaon of high resistance ssue, such as skin and bialis
muscle. AgilePulse IM is recommended for low resistance ssue
such as quadriceps muscle. Please refer to Figure 2, AgilePulse
Waveform Generator Selecon Guide, for details.
AgilePulse Waveform Generator Selecon Guide
Tissue Type Approximate
Tissue Load Range
Recommended
Waveform
Generator
Skin, intradermal
electroporaon
1100 to 2200 Ohms AgilePulse ID
Tibialis muscle 800 to 1500 Ohms AgilePulse ID
Quadriceps,
small animal
200 to 400 Ohms AgilePulse IM
Quadriceps,
large animal
80 to 250 Ohms AgilePulse IM
AgilePulse™ID System Components & Set-Up

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Front Panel
The following can be found on the front panel: the Mains/Line
power switch (illuminated when on), the Emergency Stop buon
switch, two USB ports, the ID Electrode Connector, and the User
Touch Screen (Figure 3).
One USB port can be used for a Memory Key. The data logs that
result from a vaccinaon are automacally stored on the Key if one
is inserted. The other USB port may be used for the oponal Foot
Pedal.
The Emergency Stop buon switch is used to stop system
operaon. This switch immediately stops all pulsing and turns o
the high voltage power supply. The Line/Mains power switch should
never be used to turn o the system in an emergency situaon if
pulsing is in progress.
Back Panel
On the back panel there is an IEC Power Entry module and
provisions for an Ethernet port.
Ethernet connecvity is not currently available.
Intradermal Array Electrode (IDA)
The IDA consists of two parts: the handle and the p. The handle
is connected to the waveform generator. The p contains needle
electrodes used to deliver the transfectant (DNA/vaccine/drugs
etc.) into the target cells. The needle arrays are disposable, but can
be used for mulple experiments provided proper cleaning, and
care of electrodes, are followed, and sterilized.
The IDA electrode is described in more detail on page 8.
Figure 3: AgilePulse Front Panel
AgilePulse™ID System Components & Set-Up

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Seng Up the System
The system will be set up in the following sequence:
1. Unpack the contents of the shipping box.
2. Check for obvious signs of exterior damage. If damage is
noted, contact BTX Customer Service before proceeding.
3. Place the Waveform Generator on the top of a sturdy table.
4. Connect the electrode cable into the connector at the
boom right of the front panel.
5. Connect the mains/power cord into the back panel at the
boom right.
6. Connect the foot switch (if applicable) into one of the USB
ports at the boom le corner of the Waveform Generator
(front panel).
PLEASE CONTACT BTX IF YOU ARE INTERESTED IN AN
ON SITE TRAINING.
Inial System Test
This secon will describe the process to verify mains/line cable has
been installed properly and the computer boots up.
1. Connect the system power cord to the mains.
(Plug in the device.)
2. Turn the rocker switch (Line/Main Power) on the front
panel to the “on” posion (I).
The rocker switch labeled “Power” should illuminate, and the Login
Screen should appear within 10 seconds. If the Waveform Generator
power switch fails to illuminate, then return it to the OFF posion
(O). Verify the power cord is properly plugged into the wall. Verify the
wall socket is funconal. It may be necessary to check the fuse. See
Maintenance secon (page 28) for instrucons, proper replacement
of the fuse and the correct fuse type.
If the power is properly applied, the screen should appear as that
shown in Figure 4. Aer entering the user login informaon and
tapping “OK”, the soware connues its inializaon procedure and
the NEXT buon will have a white ll.
If the internal system checks are successfully completed, the NEXT
buon will turn GREEN indicang the system is ready for use (Figure
5). If the NEXT buon turns RED, then the internal checks failed
(Figure 6). The system cannot be used if the buon is red. Contact BTX
Customer Service for assistance.
This completes the set-up and tesng of the AgilePulse™ System.
If there is a problem, contact Technical Support for assistance.
Phone [phone], or toll free 800-272-2775
Website www.btxonline.com
Figure 4: Login Screen
(visible about 10 seconds after power on)
Figure 5: System OK Screen (Operating Correctly)
Figure 6: Self Check Was Not Successful Screen
AgilePulse™ID System Components & Set-Up

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
This chapter describes the procedure to operate the Waveform
Generator and to use the Intradermal Electrode.
Experiment Delivery
The user operates the system via the front panel touch screen.
When the power is turned on, the system boots up, and displays
the opening screen. The system is immediately ready for use.
All data logs produced for a transfectant delivery will be
automacally placed on a USB Key if one is inserted into an open
slot on the front panel. The user must ensure the USB Key is
inserted before the Protocol Delivery Screen is displayed, as the
protocol pulsing is started.
If the user does not have a USB Key inserted at run me, all run
history log les can be retrieved at a later me from the log les.
USB Key/Memory Sck
The USB key can be inserted into either of the two USB ports that
are located on the front panel of the waveform generator. It should
be inserted before delivering pulses and can be removed at any
me aer the “saving data” message disappears. The USB key may
be removed without performing any addional procedures. The
data logs that result from a experiment are automacally stored on
the Key.
Intradermal Array (IDA) Electrode
The IDA Electrode consists of two parts: The handle and the p.
IDA Electrode Handle
The handle consists of an assembly into which the p is placed
and a permanently aached cable. The cable is connected to the
Waveform Generator. The handle is made of a durable plasc. It is
not sterile and may be cleaned with alcohol. The handle is reusable.
IDA Electrode Tip
The IDA p is recommended for single use and is disposable.
The p is made of medical grade plasc with surgical steel needles.
There are three ps that may be used with the handle.
The ps are shipped in individual pouches. A cover is included to
protect the needles during shipping and handling.
When ready to use the electrodes, open the pouch and remove
the p.
Insert the p into the handle by aligning the arrow on the p with
the arrow on the handle. Side grips have been added to the p for
easier applicaon.
Once the transfectant is ready for administraon, remove the
protecve cover from the IDA Tip taking care not to disconnect the
p from the handle.
System Operation
Figure 8: IDA Tip
Figure 9: Attaching IDA Tip to Handle
Figure 7: IDA Electrode Handle

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Figure 10: User Login Screen
System Operation
Waveform Generator
Login Screen — User Idencaon Input
Aer power is turned on, a Login Screen is presented that requires
the user to enter a user name and password combinaon. The
Login system is designed to prevent unauthorized users from
accessing the system and conne authorized users to permissible
acvies.
Two types of users are dened: system administrator and standard
user. There is only one system administrator of the system who has
the ability to add/remove pulse waveform protocols, add/remove
standard user accounts, and assign pulse waveform protocol access
privileges to standard users. The administrator always operates
under the “admin” account.
Standard users are intended to be everyday users of the system,
who only have the ability to run pre-dened pulse waveform
protocols from an access list as determined by the administrator.
The administrator may grant a standard user the ability to modify
an already dened protocol, but not to add/remove protocols.
AgilePulse is factory congured with only the “admin” account
available. By default no password is dened so the user can enter
the system with parameters matching that of Figure 10 on rst
log in. The password may then be changed from the User account
screen in Setup secon. The most recently logged in user will be
displayed in the User Name eld on startup.
To enter User names and Passwords, touch the Change buons to
acvate a virtual keyboard.
User name: Enter the user’s name to login
Password: Enter the password associated with the user
account. Password entry is masked with “*”
characters in order to provide increased
security.
When the User name and Password have been entered, touch OK
to log in.
Welcome Screen — Idencaon Input
An opening screen (Figure 11) allows input of key experimental
informaon aer the inial system log in. Informaon is entered
by a virtual keypad. The purpose of this screen is for the USER
(Researcher) to enter the Experiment ID, Transfectant and
Electrode ID.
All three parameters are set by user selected input. Touching a
text eld box highlights it blue and allows modicaon. Use the
available touch screen keypad to enter up to 15 characters for
each. Alternavely, a USB keyboard may be plugged into one of
the front panel USB connectors and used to edit the ID elds
manually. This screen cannot be operated from the foot pedal
(ID elds cannot be selected or edited). The following pulse
delivery screen may be operated by either the touch screen or
by the oponal foot pedal depending on the specicaon.
Aer verifying the idencaons are correct go to the delivery
screen by touching NEXT.
Figure 11: Opening Screen

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Experiment Delivery Screen Funcons
and Operaon
The purpose of the delivery screen is to iniate pulses to deliver
transfectant to ssue. The procedure is as follows.
1. The researcher injects the transfectant forming a bleb under
the skin of the animal, and inserts the electrode
according to the approved protocol.
2. The researcher determines if the electrode is inserted
properly by touching the LOAD buon or by depressing
the foot pedal. A number will appear in the Est. Load
box. LOAD buon will change to a green color. Esmated
load in ohms is displayed. This number is the eecve
resistance of the animal’s skin and will connue to be
updated every second.
Figure 12: Delivery Screen — Initialization
Figure 13: Delivery Screen — Load Estimation
3. If the LOAD Reading is within range (Less than 3500
ohms for skin) then the next step is to turn on the high
voltage power supply by touching the READY buon or
depressing the foot pedal. The READY buon will ash
green unl fully charged and then will stay solid green.
The high voltage reading will appear in the System
message box. If the researcher wants to abort the process,
the electrode should be removed and the DONE buon
pressed. In this case, an early terminaon message will
appear in the data log and the system reset.
System Operation
4. If the researcher determines that the system voltage is
correct, pulsing is inialized by touching START or
depressing the foot pedal. The START buon turns a
solid blue while pulsing is acve and solid green when
pulsing is completed.
5. When the system reaches a shutdown state either by
successful waveform delivery or by error, all buons will
turn solid green and pulse delivery data will be
displayed in the System message text box. The load
message box will change from the esmated load value
to the actual high voltage pulse monitored value.
Figure 14: Delivery Screen — High Voltage Power Supply Charge
Figure 15: Delivery Screen — Pulse Waveform Active

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
7. Press the color-lled DONE buon to return to the
opening screen.
6. All run history log data are automacally stored in
internal Flash Memory as well as a USB Key, if one is
inserted.
Figure 16: Delivery Screen — Shutdown
System Operation
Experiment Delivery Logs
All collected data from the therapeuc/vaccine delivery are
automacally stored in the internal Flash Memory and an inserted
USB key under a unique chronological directory structure.
Included items are an XML le containing raw detailed system and
runme data, a text log with basic runme data, and a comma-
separated CSV le containing pulse monitor data. The directory
structure is constructed in a “AgilePulse\Log\<Year>\<Month>\<D
ay>\<Time>” hierarchy. A maximum of 20,000 delivery logs can be
stored in the internal system memory.
Raw Data
(CCEP <Date> <Time>.xml)
Text Log
(CCEP <Date> <Time>.log)
CSV Data
(CCEP <Date> <Time>.csv)
Start and Stop Date • •
Start and Stop Time • •
Experiment •••
Transfectent •••
Electrode ID •••
Process Compleon Type • •
Set Pulse Parameters •••
Average monitor pulse voltage •
Average monitor pulse current •
Average monitor pulse resistance •
All monitor pulse voltage samples • •
All monitor pulse current samples • •
Monitor width of each pulse •••
Monitor interval of each pulse •••
Pre-pulse Load Reading • •
System Serial Numbers •
System Soware Versions •

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Figure 17: Text Log Screen
Text Log
The text log contains the same informaon that appears on the
transfectant/vaccine delivery process screen aer compleon.
Text log data is summarized in an easy-to-read format and is
designed to provide quick feedback regarding the delivery
compleon. An example Log printout is given below in Figure 17.
Field Descripon
Pulse Pulse number
VSet Voltage programmed to high voltage power supply
VMon Voltage monitored during pulse
IMon Current monitored during pulse
RMon Equivalent load resistance calculated from voltage and
current monitors R = V / I
DurSet Pulse duraon programmed to waveform generator
DurMon Pulse duraon monitored by independent
me-auditor system
IntSet Pulse interval programmed to waveform generator
IntMon Pulse interval monitored by independent
me-auditor system
Date Start: 8/14/2011 6:00:09 AM (8/14/2011 1:00:09 PM GMT)
Experiment: Pl
Transfectant: Plasmid A
Electrode: IDA-2001
Operator: BTX
Final Completion Code Code: 0 (Success)
++++++++++++++++++++++++++
Start: 8/14/2011 6:00:09 AM (8/14/2011 1:00:09 PM GMT)
Process Completion Return Code: 0 (Success)
Pulse VSet PVMon PIMon RMon DurSet DurMon IntSet IntMon
1 450 450 0.351 1282 0.050 0.050 0.200 0.200
2 450 451 0.322 1401 0.050 0.050 50.000 49.856
3 110 110 0.141 783 10.000 9.975 20.000 19.944
4 110 109 0.146 750 10.000 9.975 20.000 19.944
5 110 108 0.147 736 10.000 9.975 20.000 19.944
6 110 108 0.141 767 10.000 9.975 20.000 19.944
7 110 108 0.132 821 10.000 9.975 20.000 19.944
8 110 108 0.131 827 10.000 9.975 20.000 19.944
9 110 108 0.159 681 10.000 9.975 20.000 19.947
10 110 109 0.144 756 10.000 9.975 20.000 0.000
Load Reading: 204
Date Stop: 2/12/2007 2:16:40 PM (2/12/2007 10:16:40 PM GMT)
Time Elapsed: 00:00:18
System Operation

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AgilePulse ID In Vivo Gene Delivery System
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CSV Data
Detailed pulse informaon is exported from raw data and formaed to a standard comma separated le, primarily meant to be imported
into Microso Excel® for further user manipulaon. As an example in Figure 18, a user chart was made to display the monitored amplitude
samples of three 100 microsecond pulses run at 5 kHz. In this case, each pulse contains eight amplitude samples.
Figure 18: Sample Amplitude Analysis
System Operation

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Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Delivery Compleon Return Codes
Return codes are available following system shutdown describing how it took place.
Please contact BTX technical support to resolve the errors.
Return Code Descripon
0 Successful compleon (Normal Operaon)
1 Emergency Flash memory storage failure
2 Waveform protocol data integrity vericaon error
3 Waveform protocol mode not recognized
4Protocol voltage below minimum specicaon
5Protocol voltage above maximum specicaon
6 Protocol pulse width below minimum specicaon
7 Protocol pulse width above maximum specicaon
8 Protocol pulse interval below minimum specicaon
9 Protocol pulse interval above maximum specicaon
10 Protocol pulse number below minimum specicaon
11 Protocol pulse number above maximum specicaon
12 Protocol has a zero pulse group in between two non-zero pulse groups
13 Protocol pulse sengs exceed maximum duty cycle
14 Low voltage power supply failure (Hard system turn-o)
15 High volt power supply voltage above maximum voltage
16 High volt power supply maximum current draw
17 Pulse voltage monitor detected o-zero voltage while no pulse was acve
18 Pulse voltage monitor below maximum droop specicaon
19 Pulse voltage monitor above maximum overshoot specicaon
20 Pulse current monitor below minimum specicaon
21 Pulse current monitor above maximum specicaon
22 Independent me-audit system failed to respond to query
23 Independent me-audit system detected less pulses than expected
24 Independent me-audit system detected more pulses than expected
25 Independent me-audit system exceeded maximum measurable pulse number
26 Independent me-audit system detected excessive pulse duraon variance
27 Independent me-audit system detected excessive pulse interval variance
28 Independent me-audit system data integrity vericaon failure
29 Reset buon on front panel triggered
30 Control system watchdog meout
251 High voltage meout
252 Load esmaon meout
253 Delivery process was terminated prematurely
254 Control system experienced a system reset of unknown source
255 Load esmate outside of allowable range
System Operation

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Soware Maintenance Mode
The authorized system administrator is provided with the means
to set system access privileges, add and delete pulse protocols,
and access delivery log les. Non-administrator standard users can
modify pulse protocols if given access by the administrator and
retrieve previous experimental delivery logs. To enter this mode,
press the Setup buon on the startup screen.
User Account Maintenance
The user account maintenance screen is accessed from the
Welcome screen:
Touch SETUP
Touch USER
User accounts and privileges are managed from this screen. While
standard users will only see the Acve User secon, and thus will
only be able to change their password, the system administrator
has access to account properes for all standard users.
As an administrator, a new user account can be adding by pressing
the “Add New User” buon. Once the account is created the
new user is assigned read-only access to all of the pulse protocol
waveforms on the system. The administrator may then select
individual protocols and provide more access by giving both read
and write privileges, or they may restrict access to a protocol
altogether by giving the user no access at all. When a user is not
given any protocol access they will not be able to see or select it
from the Protocol screen when they log in under their own user
name. When protocols are added through the Protocol screen aer
a standard user account has already been created, all user accounts
in the system will receive read-only access to the new protocol.
The administrator also has the ability to change a user’s password.
This is ideal for situaons in which the user forgets their password
and thus need the password to be reset in order to log in again.
Caution
IMPORTANT! The changes made on this screen
will only be permanently saved when the HOME
button is pressed to go back to the Welcome
Screen. If modications are made and system
power is turned off while still viewing the screen,
all changes will be lost.
Figure 19: User Account Screen
System Operation

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Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Log Retrieval
All previous experimental delivery logs can be retrieved and saved
onto a USB Key or deleted from internal memory. The interface
consists of a series of chronological sub-folders from which the user
is allowed to double-tap on the touch screen to descend through
the hierarchy. The standard naming sequence is Year, Month, Day,
followed by special folders with the me of day. These me folder
icons show a hand underneath, meaning they contain all les
generated from a single vaccine/transfectante delivery. The “ ”
folders allow the user to return to the previous level of hierarchy.
Special me folders display basic informaon on the right hand
side of the screen when selected. Idencaon numbers, me of
day, and status compleon indicators help the user nd the correct
vaccine/transfectant delivery run to download.
When selected by a single tap on the touch screen icon, any
level of hierarchy can be saved to a USB Key inserted in the front
panel by pressing the SAVE buon. The hierarchy structure will
be maintained and copied to the USB Key. The DELETE buon
is also available to remove the hierarchy from internal memory.
This delete is permanent and the lost les cannot be recovered.
A maximum of 20,000 delivery logs can be stored on the internal
system memory.
DEPENDING ON THE NUMBER OF FILES TO BE DELETED, THIS
PROCESS CAN BE SLOW.
Figure 20: Log Retrieval Screen
System Operation
Pulse Protocol
The pulse protocol to be delivered at run me is set by pressing
the PROTOCOL buon and adjusng parameters via the touch
screen interface. Parameters for three pulse groups are modiable
by selecng a box and scrolling through the available values.
Subsequent groups are acvated by selecng more than one pulse
in the number eld and conversely deacvated by selecng zero
pulses in the number eld.
The adjusted protocol is saved to internal memory when the HOME
buon is pressed. Minimum and maximum parameter values are as
follows:
Protocol Parameter Boundaries
Amplitude (volts) Duraon (ms) Interval (ms) Pulse # Pulse Groups
Minimum 50 0.050 0.200 (5 kHz) 1 1
Maximum 1000 10* 1000 (1 Hz) 10 3
* For Pulse Amplitudes greater than 300 V, the maximum allowable Pulse Width is 1 ms
** Pulse duty cycle is limited to 50%
Caution
IMPORTANT! The changes made on this screen will only be permanently saved when the HOME button is pressed
to go back to the Welcome Screen. If modications are made and system power is turned off while still viewing the
screen, all changes will be lost.
Figure 21: Pulse Protocol Modication

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AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
System Name Company Mechanism Target Factors
(All pain rangs are esmates.)
AgilePulse BTX Inject transfectant in dermis
or muscle with hypodermic
needle, insert needle array,
apply electric eld to deliver
into cells
Dermis/Tibialis muscle • Large dose
• Reduced pain compared to
other electroporaon systems
• Pain 3 out of 10
• High cell transfecon
PMED™PowderMed Microscopic parcles coated
with DNA, delivered into cells
using compressed helium
Epidermis • Small doses
• Possible taooing
• Pain 3 out of 10
• Low/Medium cell transfecon
Biojector® 2000 Bioject Medical
Technologies
Inject using CO2 pressure with
spacer (subcutaneous) or
without spacer (to muscle)
Skin or Muscle • Large doses
• Pain 3 out of 10
• Medium cell transfecon
ECM 830,
Gemini X2,
and others
BTX and others Inject into muscle insert
electrode and apply electric
eld
Muscle • Large dose
• Signicant pain
• Pain 5 to 6 out of 10
• High cell transfecon
Many Many Deliver viral vector with
hypodermic needle into
muscle
Muscle • Immune system reacts to viral
vector, inecient when boosng
• Pain 2 out of 10
Applicaons
The AgilePulse™ system is used to deliver molecules of interest to skin or muscle ssuesn for research purposes only. The AgilePulse system
has been shown to increase DNA delivery to cells resulng in 100 to 1000 mes higher protein expression compared to hypodermic needle
injecon alone (Roos et al., 2006).
The AgilePulse system is typically used to deliver cancer vaccines in doses of 50 µg or more. The AgilePulse system includes a AgilePulse
voltage waveform generator and an Intradermal Array (IDA) electrode used to convert the voltage to electric elds which deliver the
vaccine directly into cells in the skin.
Transfectant Delivery Methods
There are several methods used to deliver transfectants of interest.
AgilePulse™ID Tutorial

18
AgilePulse ID In Vivo Gene Delivery System
Publicaon 015-101444 Rev 2.0 • www.btxonline.com
Ions
Gene
Cell
Membrane
Before Field Applied
Field Applied-Ions Move
Field Applied-Pathways Form
Field Removed-Membrane Seals
Figure 22: Electroporation
Delivery Into Cells Using
Electroporaon
General Electroporaon Discussion
Electroporaon is the use of a transmembrane electric eld pulse
to induce microscopic pathways (pores) in a bio-membrane. Their
presence allows molecules, ions, and water to pass from one
side of the membrane to the other. As the right side bar shows,
when the electric eld is applied, the ions inside and outside the
cell membrane migrate. As the charge builds up on either side of
the membrane, the membrane weakens and the pathways form
perming material outside of the cell to enter. If the electric
eld is promptly removed the pathways close and the membrane
reseals. If the electric eld duraon is too long the pathways
increase and the cell is killed. Ecient electroporaon depends on
proper selecon of electric eld waveforms. The electropores are
located primarily on the membrane areas which are closest to the
electrodes. The pathways form in about a microsecond and seal
in seconds to minutes. The duraon of the electric eld is tens of
microseconds to tens of milliseconds.
The use of electroporaon was described by Neumann in the early
1980s. The roune use of electroporaon became very popular
with researchers through the 1980s because it was found to be a
praccal way to place drugs, or other molecules into cells. In the
late 1980s, sciensts began to use electroporaon for applicaons
in mul-cellular ssue.
In the early 1990s Lluis Mir of the Instute Gustave-Roussy was
the rst to use electroporaon in a human trial to treat external
tumors.
Research has shown that the inducon of pathways is aected by
three major factors. First, cell-to-cell biological variability causes
some cells to be more sensive to electroporaon than other
cells. Second, for pathways to be induced, the product of the
pulse amplitude and the pulse duraon has to be above a lower
limit threshold. Third, the number of pathways and eecve
pathway diameter increases with the product of “amplitude” and
“duraon.” Although other factors are involved, this threshold
is now understood to be largely dependent on a fourth factor,
the reciprocal of cell size. If the upper limit threshold is reached
pore diameter and total pore area are too large for the cell to
repair by any spontaneous or biological process. The result is
irreversible damage to the cell or cell lysis. Because the mechanism
of electroporaon is not well understood, the development of
protocols for a parcular applicaon has usually been achieved
empirically, by adjusng pulse parameters (amplitude, duraon,
number and inter-pulse interval).
AgilePulse™ID Tutorial