4D-Nucleofector® Manual

For Research Use Only
4D-Nucleofector® Manual
The purchase of the 4D-Nucleofector® conveys to the buyer the non-transferable right to use the system as well as Lonza’s proprietary
Nucleofection® Technology for research conducted by the buyer (whether the buyer is an academic or for-profit entity). For further details
about the license please refer to chapter 5.0.
1
Contents
1.0 The Amaxa® Nucleofector® Technology
3
2.0 Operating Instructions
5
2.1
Restrictions
5
2.2
Maintenance
5
2.3
Safety Instructions
7
2.4
Waste Disposal
7
2.5
4D-Nucleofector® System Components
8
2.6
System Set-up Instruction
9
2.7
Instructions for Using the 4D-Nucleofector® System
2.7.1 Turning on the 4D-Nucleofector® System
2.7.2 Adjusting position of the graphical user interface (touch screen)
2.7.3 Operation software
2.7.4 Using a predefined experiment
2.7.5 Defining an experiment
10
10
10
11
12
13
2.8
Additional Settings and Options
14
2.9
Cleaning the Nucleocuvette® Vessel Tray
15
2.10 Firmware Update
3.0 Troubleshooting
15
16
Contents
2
4.0 Error Codes of the 4D-Nucleofector®
18
5.0 Purchaser Notification
21
6.0 Appendix
23
3
1.0 The Amaxa®
­Nucleofector®
­Technology
Since its introduction in 2001, Amaxa® Nucleofector® Technology
has transformed transfection. Nucleofection® has proven to be a
reliable and reproducible easy-to-use technology, suited to a wide
variety of applications. An ever increasing number of publications
in a wide range of research areas reflect how Nucleofector®
Technology is driving research in numerous cell types and
applications. Nucleofector® Technology provides an answer to
almost every transfection challenge.
Transfection of any cell
With over 160 protocols optimized by Lonza’s R&D team for cell
lines and primary cells and more than 680 entries in the online
cell database, Nucleofector® Technology is clearly the transfection
method-of-choice for any difficult-to-transfect cell type. Moreover,
Nucleofection® is the only electroporation based technology
allowing reliable transfection of adherent cells and cells in
suspension.
How it works
Nucleofector® Technology is based on two unique components,
the Nucleofector® System that delivers the specifically optimized
electrical parameters and Nucleofector® Kits, which contain specif­
ic Nucleofector® Solutions. As part of Nucleofector® Technology,
Lonza provides cell-type specific Optimized Protocols for many
different cell lines and primary cells.
Transfection of any substrate
Nucleofector® Technology offers high flexibility within applications,
since the same transfection parameters apply for almost all
substrates. DNA vectors, RNA duplexes, and peptides can be
transfected using our sophisticated transfection protocols. This
makes Nucleofection® an ideal tool for providing answers to
scientific questions in over-expression studies, gene silencing
approaches, protein expression, generation of stable clones and
many more applications besides.
The Amaxa® Nucleofector® Technology 1.0
4
Moving transfection into the fourth dimension
At Lonza, we believe that listening to our customers is the best
way to develop innovative products that meet your needs. In line
with this customer-oriented spirit, the 4D-Nucleofector® System
was developed to offer advanced performance, flexibility and
convenience. The system is controlled by intuitive software on an
up-to-date touch screen user interface. Its modular architecture
allows seamless expansion of the system for new applications.
Experience the new 4D-Nucleofection® with:
—— A novel Conductive Polymer based cuvette system free of
toxic metal ions
—— Transfection of different cell numbers using the same
conditions
—— Transfection of adherent cells in using multiple methods
4D-Nucleofector® System
Figure 1: 4D-Nucleofector® with consumables
Support Teams:
EU
Phone: +49 221 99199 400
E-mail: [email protected]
North America
Phone: +1 800 638 8174
E-mail: [email protected]
5
2.0Operating Instructions
2.1
Restrictions
Medical use restrictions
Nucleofector® Technology is intended for research and investi­
gational use by professionals only. Please note that Lonza’s
Nucleofector® Technology is not intended to be used for diagnostic
purposes, for testing or treatment in humans.
License statement
Lonza is holder of various patents, patent applications, copyrights and technical and scientific experience with respect to
Nucleofector® Technology. Use of Lonza’s Nucleofector® Technology
and/or related software requires a license from Lonza.
Purchasers are granted a non-exclusive, non-transferable license
for a limited use of Lonza’s Nucleofector® Technology and related
software for research purposes, the terms of which are disclosed
in detail in chapter 5.0. Certain commercial application is allowed
under Lonza’s license for for-profit-entities.
For license information contact Lonza Cologne AG by phone
+49 221 99199 0 or e-mail: [email protected]
2.2
Maintenance
The 4D-Nucleofector® System requires minimal maintenance for
reliable operation. Unplug the power supply before cleaning and
disinfecting the case. Use a damp cloth to wipe down the outer
case with water or 70–80% ethanol. Avoid wetting the cuvette
holder within the cuvette tray and the connectors located on the
rear of the system.
The 4D-Nucleofector® System is designed for use under a sterile
hood either with or without a UV radiation source. Prolonged
exposure of the outer casing to UV light may lead to discoloration
but not functional impairment of the Nucleofector®. If sterile
hoods are sterilized on a regular basis by overnight UV irradiation,
we recommend protection of the system by appropriate shielding.
The system should be removed from the hood during extended
UV irradiation.
The 4D-Nucleofector® is protected by two main fuses. Both are
inside a receptacle incorporated in the power socket of the Core
Unit (see Fig. 2.2, page 8). Blown fuses can easily be replaced.
Disconnect the 4D-Nucleofector® from the power supply and
insert a small flat screwdriver into the slot on top of the power plug
housing to lever it open. The fuse receptacles (each marked with
an arrow) are then also levered open using the screwdriver.
Operating Instructions 2.0
6
Figure 2.0: Opened fuse receptacles
The 4D-Nucleofector® will not function unless both the left and
right receptacle contain intact fuses. Blown fuses can usually
be identified by molten broken wires inside the glass tube. Only
use T2.5A, L250V fuses to replace blown fuses.
7
2.3
Safety Instructions –
please read carefully
This symbol means that there is a risk of electric shock. An electric
shock could cause death or personal injury.
The 4D-Nucleofector® System has been certified by international
safety standards and is safe to use when operated in accordance
with this manual.
This system is designed to deliver variable high voltage electrical
impulses for the purpose of introducing substrates into eukaryotic
and prokaryotic cells.
These electrical impulses can be DEADLY!
Therefore, use this system with care and take the following
PRECAUTIONS:
—— Only use the system once you have read the 4D-Nucleofector®
Manual. The manual should be accessible for all users. Make
sure that each potential user reads it.
—— Do NOT open the system. The system does not contain userserviceable parts. Under NO circumstances should circuit
components be interfered with, as they can deliver an electric
shock even when system is not in operation.
—— Do NOT alter the system in any manner.
—— Do not obstruct the movement of the drawer holding the
Nucleocuvette® Vessels or culture plates.
—— Do NOT plug any non-Lonza-certified connectors into the
external high voltage electrode sockets at the rear side of
the 4D-Nucleofector® X Unit. They are designed to connect
certified add-on systems to the 4D-Nucleofector® System,
e.g., the 96-well Shuttle®.
—— Only use the system when it is set on top of a safe, level and
stable table or bench.
—— Set up the system in such a way that easy removal of the
power cord is possible at any time.
—— Do not expose the system to a humid environment.
—— The system must not be exposed to direct sunlight nor be
placed in a hot environment.
—— The system is not approved for use in fire- or explosionen­
dan­gered areas, nor for use with inflammable or explosive
media.
—— Take precautions against severe impacts and vibration while
moving and transporting the 4D-Nucleofector® System.
—— Only use the system with Lonza-certified Nucleofector®
Solutions and Lonza-certified Nucleocuvette® Vessels or
electrodes. Using consumables from any other source than
Lonza will preclude all warranty and liability claims.
—— Standard Nucleofector® Solutions and the Nucleofector®
Solutions used for the 4D-Nucleofector® System are not
com­patible. Do not use standard Nucleofector® Solutions
with the 4D-Nucleofector®.
—— Unpack the Nucleocuvette® Vessels or electrodes immediately before the experiment. Make sure that the outer contact
areas are dry.
—— Safety may be compromised if any fluid has been spilled in
the close vicinity of the 4D-Nucleofector® System. Ensure
that no fluid is in contact with or has entered the system.
—— System safety may be compromised if any fluid enters the
system. Contact Lonza Scientific Support for advice on actions
or precautions that should be taken.
—— Do not insert or place foreign objects in the electrical drawers
of the 4D-Nucleofector® System.
—— Safety may be compromised if any foreign object has
entered the 4D-Nucleofector® System. Contact Lonza
Scientific Support for advice on actions or precautions that
should be taken.
—— If the 4D-Nucleofector® System has been damaged, ensure
that the system can not be used by any personnel and
contact Lonza Scientific Support for assistance.
—— All service shall be performed by Lonza authorized personnel
only.
—— Handling of system parts that may be contaminated with
sample should always be performed using protective
gloves and any disposal of such parts must be according to
Federal, state or local procedures for clinical waste handling
and disposal. Use secure leakproof containers and avoid
unprotected handling of such parts.
Lonza disclaims all warranties and shall in no event be liable for
any kind of damages caused by or arising out of any operation or
use in violation with the above safety and handling instructions.
2.4
Waste Disposal
Disposal of consumables from Nucleofector® Kits (cuvettes,
pipettes and Nucleofector® Solutions): Dispose of cuvettes,
pipettes and expired Nucleofector® Solutions in a biohazard
container. Refer to your local waste management organization and
to the relevant laboratory safety instructions for proper disposal
practices.
Operating Instructions 2.0
8
2.5
4D-Nucleofector® System
Components
By definition the 4D-Nucleofector® System is a modular system
offering maximum flexibility. Therefore, a complete system may
vary in the number and type of components it contains. A typical
4D-Nucleofector System includes:
1
2
One 4D-Nucleofector® Core Unit including two side panels
At least one Functional Unit, including two side panels
X Unit: Comprising two retainers for 100 µl Nucleocuvettes
and one retainer for 16-well Nucleocuvette® Strips
3 Power cable
4 HV/data cable
—— 4D-Nucleofector® Manual
1
5
6
7
8
Core Unit
X Unit
Side panels
Tray holding retainers for two 100 µl Conductive Polymer
cuvettes and one 16-well Nucleocuvette® Strip
9 Touch screen (graphical user interface of the system)
10 USB port (for USB stick)
11 Operation LED
6
9
2
8
11
5
7
10
Figure 2.2: Assembled 4D-Nucleofector® System (front view with
open drawer)
3
4
Figure 2.1: 4D-Nucleofector® System before assembly (Core Unit
[left] and X Unit [right] with attached side panels, power cable
and connector)
12
13
14
15
16
17
Power cord socket with main switch and fuses
USB port (for PC connection)
Serial port for Shuttle connection
Lonza Interface outlet to connect Functional Units
Lonza Interface inlet to connect Functional Units
Lock and release slider for attachment of the Core Unit
functional modules or side panels
18 HV contacts for the 96-well Shuttle
12
14 13 15 17
18
16
Figure 2.3: Assembled 4D-Nucleofector® System (rear view)
The 4D-Nucleofector® System comes with default programs and
includes a USB port at the front for software updates.
9
2.6
Set-up Instructions
1
Unpack the components of the 4D-Nucleofector® System and
check for completeness.
2
The 4D-Nucleofector® System components (Core Unit and
Functional Units) can be assembled side-by-side or stacked
on top of each other.
—— Move both units towards each other until the housing lids
touch and attach the units to each other by pushing the lock
and release slider towards the front of the system. Units are
properly connected when the lock and release slider clicks
into place.
Locked
2a Side-by-side assembly
—— Place the Core Unit and the Functional Unit(s) next to each
other as shown in Figure 2.4, pull back the lock and release
slider, and detach side panels by gently pulling.
Figure 2.6: Rear view of attached units, with close-up of slider
—— Attach side panels to the right- and left-hand side of the
system and lock them into place as described above.
Figure 2.4: Core Unit und Functional Unit side-by-side
Unlocked
—— Make sure that the positioning bolts of one unit are properly
aligned with the appropriate slot in the lock and release slider
of the second unit. Make sure that the lock and release slider
is pulled back as far as possible.
Positioning bolt
Locked
Figure 2.5: Positioning bolts and lock and release slider
Figure 2.7: Rear view with mounted side panels
Operating Instructions 2.0
10
2.7
2b Stacked assembly
—— Unpack the units of the 4D-Nucleofector® System. Do not
remove the side panels.
—— Stack the units with the Core Unit (containing the touch
screen) on top.
Instructions for Using
the 4D-Nucleofector® System
This section gives an overview of the 4D-Nucleofector® System
operating software. Details of the Nucleofection® process can be
found within the dedicated Optimized Protocols delivered with the
4D-Nucleofector® Kits.
2.7.1
Turning on the 4D-Nucleofector® System
Turn on the system using the main power switch at the rear of the
Core Unit. The system will boot up — this process may take a few
moments — and the blue LED at the front of the Core Unit will be
lit. Once the start-up procedure is complete, the 4D-Nucleofector®
Graphical User Interface (touch screen) will display the software
main screen. The main screen displays the current configuration
of the 4D-Nucleofector® System (Core Unit and Functional Units).
Figure 2.8: Stacked units
3
Connect the Core Unit’s Lonza Interface outlet port with
the Lonza Interface inlet port of the Functional Unit below
or beside it using the Lonza Interface cable. Make sure that
the cable is securely attached and that the retaining screws
on the cable housing are screwed tightly into the unit port.
Further Functional Units are added by connecting the outlet
and the inlet ports of adjacent units as described above.
Plug in the Lonza Interface terminator cap into the outlet of
the last unit.
Figure 2.9: Lonza Interface cable connecting Core Unit and X Unit
4
Attach the power cord to the power socket at the rear side of
the Core Unit and plug it into an appropriate power outlet.
5
Check all connections before turning on the system for the
first time.
Figure 2.10: Software main screen
11
2.7.2
Adjusting position of the graphical user
interface (touch screen)
The touch screen of the 4D-Nucleofector® System can be set at
four angles (0°, 30°, 45° and 60°), providing convenient access
for the user. Press the mark on top of the display frame to unlock
the display from its home position. From this starting position,
the user can adjust it to the most convenient angle by simply
pulling it into a more upright position. The screen can be returned
to its home position by moving it to the 60° position and then
pulling it gently forwards towards the front of the Core Unit. A
switch will be activated, and the screen will drop automatically
back into the starting position. Press down the screen to lock it
in its home position.
1
2
3
4
2.7.3
Operating software
The 4D-Nucleofector® is operated via a 5.7 inch touch screen
display and controlled by intuitive software. This chapter will
familiarize you with the functions of the touch fields and the basic
features of the operating software. You will learn how to use a
predefined experiment, how to start a Nucleofection® Procedure
and how to create a new experiment from scratch.
1
2
3
Figure 2.12: Software screen elements
Home icon
5
Figure 2.11: Releasing the touch screen
(1) Return to the main menu
Top bar
(2)
Return to the “Choose a vessel” screen
Wrench icon
(3)
Open service menu
BACK
Return to previous screen
CLR
eset settings for an experiment or a
R
Nucleocuvette® Vessel
SAVE
Save experiment or results file
LOAD
Load predefined experiments
OK/START
Confirm selection or execute a program
EXPERIMENT
efined Nucleofection® Parameters for one
D
or several Nucleofection® Experiments
CELL TYPE PROGRAM
S elect the Nucleofection® Program by
cell type
CUSTOM PROGRAM
Select customer defined program
PULSE CODE
Modify pulse code
SOLUTION
isplay Nucleofector® Solution required
D
for the cell type
VOLUME
Working volume of the vessel selected
CONTROL OPTIONS
efine negative controls
D
(no DNA, no pulse)
A-Z, Z-A, Top 10,
Last 10
S ort a list alphabetically, display the
most frequent 10 items or the most
recent 10 items
Magnifier symbol
Activate search functions
i
Display additional information (text)
<,>
A rrow keys to switch between
experimental setup screens
Operating Instructions 2.0
12
2.7.4
Using a predefined experiment
This chapter summarizes the steps required to load and perform
a predefined experiment. More detailed information for defining
experiments is given in chapter 2.7.5.
—— Select the Functional Unit you wish to work with by pressing
the appropriate icon (1).
—— A screen appears displaying the Nucleocuvette® Vessels
available for the selected Functional Unit (2).
—— Press the icon displaying the vessel you wish to use.
—— A screen appears with the message
“Choose Experiment or Position” (3).
—— Press on the touch field labeled “Choose Experiment”.
—— A list of predefined programs will appear (4).
—— Choose the experiment you wish to use and confirm your
selection by pressing “OK”.
—— By pressing on the icons displaying the Nucleocuvette®
Vessels you can check the settings (Cell Type Program,
Pulse Code etc.) for the selected well (5).
—— To accept all settings, press “OK”.
—— A summary of the experimental settings will appear (6) and
the drawer of the selected Functional Unit will open, giving
access to the vessel retainers. At the same time, a message
“Load Samples” will be displayed on the touch screen.
—— If the experimental setup is as desired, load the samples and
press “START” to run the experiment.
—— Depending on the number of samples you wish to process
the drawer will be opened several times and a message
“Please change cuvette(s). Proceed?” will be displayed (7).
—— Load samples and press “YES” to continue or press “NO” to
interrupt the experiment.
—— When the experiment is complete, a result file summarizing
the Nucleofectionn® Process will be displayed.
—— By pressing “START” again a message will appear asking
“Do you want to pulse the same experiment again?” (8).
—— Press “OK” to start the experiment, “NEW” to return to the
“Experiment” screen or “CANCEL” to return to the “Results”
screen.
1
2
3
4
5
6
Load Samples
7
8
Figure 2.13: Using a predefined experiment
13
2.7.5
Defining an experiment
This chapter explains how to define and save a new experiment.
By definition, an experiment is a set of Nucleocuvette® Vessels
with defined Nucleofection® Parameters (Cell type or Custom
Program, Pulse Code and Control Options) for each vessel.
—— Select the Functional Unit you wish to work with by pressing
the appropriate icon (1).
—— A screen appears displaying the Nucleocuvette® Vessels
available for the selected Functional Unit (2).
—— Press the icon displaying the vessel you wish to use.
—— A screen appears with the message “Choose Experiment or
Position” (3).
—— Press one of the Nucleocuvette® Vessel position (e.g. A1).
—— The fields “CELL TYPE PROGRAM”, “CUSTOM PROGRAM”,
“PULSE CODE”, and “CONTROL OPTIONS” will be activated (4).
—— Press the field “CELL TYPE PROGRAM” to choose pre­
defined Nucleofection® Conditions from a cell type list.
(5) Use the search (magnifying glass symbol) or the
sort list functions (A-Z) to find conditions more quickly.
—— Select the desired cell type by tapping on the appropriate
line of the cell list. The cell type selected will be
highlighted. For additional information about the cell type
selected press “i”. To confirm your selection press “OK”.
—— If required, modify pulse code by pressing the letter or
number code fields. A keyboard will appear, enabling you
to change settings.
—— Define control options for the selected vessel by
choosing:
—— Standard: Nucleofection program applied
to vessel containing cells and
substrate
—— No Substrate: Negative control. Nucleofection®
Protocol applied to vessel with
cells but without substrate
—— No Program: Negative control. No
Nucleofection® Program applied
to a vessel containing cells and
substrate (6)
—— Save your experiment by pressing the “SAVE” button. (7) A
keyboard will appear allowing you to define a name (max.
length: 26 characters). You may enter further information
about your experiment by touching the “Info” field and typing
in your text (8).
—— Confirm and save the experiment parameters by pressing
“OK” or “SAVE”.
—— Load samples and start your experiment by pressing
“START”.
1
2
3
4
5
6
7
8
Figure 2.14: Defining and saving an experiment
Operating Instructions 2.0
14
2.8
Additional Settings and Options
This chapter explains how to change settings like date and time
and how to organize and manage your stored experiments, results
and custom programs. It also gives more information about the
update functions of the 4D-Nucleofector®.
—— Activate the “Settings” menu by either pressing the “Core
Unit” icon or the “Wrench” icon submenu “SETTINGS” (1).
—— A screen appears displaying a list of setting options (2):
—— Experiments:
Display user-defined experiments
—— Custom Programs:Create and manage custom
Nucleofection programs
—— Results:Load result files onto a USB stick
plugged into the USB port in front
of the Core Unit
—— Lonza Programs:Version information about the cell
type list
—— Display and Audio:Adjust brightness of the touch
screen display, time to activate
standby and deactivate the
touch tone
—— Date and Time:
Set and format date and time
—— Language and:
Change language settings for
keyboard
display functions and keyboard
—— . . . more:To switch between pages of the
settings list
—— Firmware Update: Update the system software
(see chapter 2.10 for details)
—— System Restart:
Restart system
—— Device Cleaning: Opens the tray allowing to remove
it for cleaning (see chapter 2.9)
To open one of the setting menus touch the appropriate field.
Depending on the menu, several parameter settings will appear.
Most of the menus are intuitive. The menus requiring additional
information are:
Experiments
This menu displays a list of experiments and allows you to view
additional information by pressing the “i” field. Erase existing
experiments by pressing “CLR”. To load a predefined experiment
from a USB stick, insert the stick into the USB port on the front of
the Core Unit, press “LOAD” and follow the instructions displayed
on the screen (3).
Results
This menu will display a list of result files and allows you to manage
them. To erase result files that are no longer required, select the file
from the list and press “CLR”. To store result files on a USB stick,
insert the USB device into the USB port on the front of the Core Unit
and press “SAVE”. Pressing “i” displays further information related
to the result file (4).
Custom Programs
This menu is used to create and manage custom Nucleofection
programs. You can create new custom programs by pressing the
“NEW” field. Edit existing programs by pressing “EDIT” or save your
programs onto a USB stick by pressing “SAVE” (5).
To create a new custom program or to edit an existing program:
—— Press “NEW” or select an existing program and press “EDIT”.
—— A screen will open comprising a keyboard, “PULSE CODE”
setting, a “SOLUTION” field and a “VOLUME” selection.
—— Define a name for your custom program by typing, e.g., the
cell name.
—— Select the volume appropriate for the Nucleofection vessel
you wish to use (20 µl for the 16-well Nucleocuvette strip or
100 µl for single Nucleocuvettes).
—— Press “SOLUTION” to display a list of available solutions.
—— Select the solution most suited for your cell from the list and
confirm your selection with “OK”.
—— Define the Nucleofection program by typing in the “PULSE
CODE” — you can determine the best program by using one of
our optimization kits or simply use the code from an existing
Optimized Protocol. (6)
—— Confirm the setting for your custom program by pressing
“OK”.
—— The custom program can now be selected for an experiment
by pressing the “CUSTOM PROGRAM” field (see chapter 2.7.5
for further details).
15
2.9
1 Activate “Settings”
3 Menu “Experiments”
2
4 “Results”
Cleaning the Nucleofection
Vessel Tray
To allow convenient cleaning, the Nucleofection Vessel Tray can be
removed from the X Unit. To dismount the tray please follow the
instructions below:
—— Open the “SETTINGS” menu as described in chapter 2.8.
—— Press “DEVICE CLEANING”.
—— The drawer will open, giving access to the Nucleofection
Vessel Tray.
—— Remove the tray from its support by pressing the two
plastic noses on the left and right side of the tray.
—— Wash the tray with a cloth damped in water and rinse it
with tap water until all leftovers are washed off.
—— Dry the tray carefully.
—— Remount the dried tray to the support.
—— Press “DONE”.
—— The drawer will close and the tray will be moved to its
home position.
2.10 Firmware Update
5 Menu “Custom Programs”
6 Menu “Custom Programs”
Figure 2.15: Additional settings and options for organizing
stored experiments
Due to new Optimized Protocols added to the Lonza Program
list or technical improvement of the operation software, the
4D-Nucleofector® may require an update of its firmware. To update
the firmware proceed as follows:
—— Download the software from the Lonza web site (see Appendix
for further details) and store the file(s) on a USB stick into a
folder called “LONZA”.
—— Start the 4D-Nucleofector® System.
—— The “Choose a device” main screen will appear.
—— Click on the Core Unit icon.
—— Select “Firmware Update” within the “SETTINGS” menu
—— A dialog box will appear asking to plug in the USB stick.
—— Plug in the USB stick and press “OK”.
—— Follow the instructions on the screen to update the firmware.
Operating Instructions 2.0
16
3.0Troubleshooting
The following troubleshooting guidelines may be helpful if
experiments using the 4D-Nucleofector® System do not provide
the expected results. The comments are intended to help optimize
experimental conditions.
If you require further help, please contact your local Scientific
Support Team.
Issue
Low survival rate
Possible error
Cells were kept in Nucleofector
solution too long.
Cells were damaged by harvesting
procedure or through handling.
Cells culture conditions were
suboptimal.
Multiple use of cuvettes.
Poor DNA quality.
Europe
Phone +49 221 99199 400
Fax +49 221 99199 499
[email protected]
www.lonza.com/nucleofection-support
North America
Phone 800 521 0390 (toll free)
Fax +1 301 845 8338
[email protected]
www.lonza.com/nucleofection-support
Solution
Transfer cells immediately into pre-warmed medium as recommended in the
Optimized Protocol.
Avoid harsh conditions during cell harvesting, especially centrifugation at higher
speed or overexposure to trypsin.
Pipette cells smoothly as they are quite stressed already. Use a plastic pipette as
recommended in the Optimized Protocols.
Cells should be viable and in culture for several passages. Avoid excessive
cell densities or cell confluencies since this may decrease cell viability post
Nucleofection. For further details please refer to the dedicated Optimized Protocol.
We strongly recommend using the Nucleofection vessels only once, because the
high voltage pulses that are applied drastically affect their physical integrity.
DNA used for Nucleofection should be of high purity. We strongly recommend
endotoxin-free preparation of the DNA. Do not use procedures involving phenol/
chloroform treatment.
17
Issue
Low substrate
transfer
Possible error
DNA amount is too low.
Cell number in Nucleofection
sample too high or too low.
Poor DNA quality.
Solution
We recommend a DNA amount of 0,2–5 µg per sample. If both transfer efficiency
and cell mortality are low, the DNA amount could be increased up to 10 µg per
sample. Increasing DNA amount may lead to higher transfection efficiency, but
at the same time result in higher cell mortality. For further details please see
dedicated Optimzed Protocol.
Please use the cell numbers recommended in the dedicated Optimized Protocol.
DNA used for Nucleofection should be of high purity. We strongly recommend
endotoxin-free preparation of the DNA. Do not use procedures involving phenol/
chloroform treatment.
Troubleshooting 3.0
18
4.0 Error Codes of the
4D-Nucleofector®
The following table lists Nucleofector® error codes and provides
suggestions for solving the problem. If the problem cannot
be resolved please call Lonza’s Scientific Support Team
(Europe/World: +49 221 99199 400; USA: 800 521 0390).
Alternatively, use our internet and e-mail based scientific support
at www.lonza.com/nucleofection-support. If the 4D-Nucleofector®
System must be returned for repair, please contact our Scientific
Support Team for shipping and warranty instructions.
Arc discharge correction
Arcing is a complete or partial discharge circumventing the sample
and is often accompanied by a flash and a noise. This problem
is usually caused by imperfect cuvettes or cuvette filling. The
4D-Nucleofector® System is equipped with a hardware safety
feature that immediately detects arc formation and protects the
cells from damage. After the arc interruption the 4D-Nucleofector®
System resumes program execution. Normally, the program
can be completed successfully (“arc discharge correction
1 or 2”) and only minor differences in transfection efficiency
are observed. When repeated arc discharges occur within one
program, it may be impossible for the 4D-Nucleofector® System
to complete execution of the program. In this case (“Err8 – Arc
discharge”), significant impact on transfection efficiency may be
observed. Discard the cuvette (which is possibly damaged) and
its contents, reset the device by pressing any button and repeat
the experiment with a new cuvette. It is not necessary to switch
off the 4D-Nucleofector® System.
Color Code of Wells
Parameter file
Undefined
Defined
No program control
No DNA control
Result file OK
Result file Error
19
Error code
What happened?
Possible error
Procedure
Err 1A
No pulse generated.
Internal communication failure,
internal error or device possibly
defective.
Clear the error message by
pressing any button and try to
apply program a second time. If
Err1A re-occurs, switch off the
device, check the LONZA Interface
cable, wait for 2 seconds and
switch on again. If error persists,
contact our Scientific support.
Err 1B
Pulse generated with unclear
outcome.
Internal communication failure,
internal error or device possibly
defective.
Clear the error message by
pressing any button and use
sample if maximum efficiency is
not essential. Otherwise, try with
a new sample. If Err1B re-occurs,
switch off the device, wait for
2 seconds check the LONZA Inter­
face cable, and switch on again.
Err 2
No pulse generated.
No cuvette or inappropriate
cuvette loaded. Inappropriate
Nucleofector® Solution or volume.
Check cuvette. Check type and
volume of Nucleofector® Solution.
Cuvette can be reloaded.
Err 3
No pulse generated.
Improper interconnection or
system defective
Switch off the device. Check
proper connection of the Lonza
Interface cable as well as the
terminator cap on the last Lonza
Interface Outlet. Switch on the
device and repeat the experiment
with the same sample.
Err 4 WEAK
Re-used cuvette or inappropriate
Nucleofector® Solution.
A weak pulse occurred.
Efficiency may be sub-optimal.
Clear the error message by
pressing any button. Use the
sample if maximum efficiency is
not essential. Otherwise, use a
new sample.
Err 5 & Err 6
Device possibly generated an
arc discharge. Program could
be resumed and successfully
completed. A limited impairment
on performance has to be
presumed.
Inappropriate solution or well
volume, occasional arcing.
Utilize sample if maximum
performance is not essential.
Check solution and volume.
Err 7
No pulse generated.
No lid detected on the cuvette.
Clear the error message by
pressing any button. Check
proper placement of the lid and
rerun experiment with the same
HC LC
cuvette.
Error Codes of the 4D-Nucleofector® 4.0
20
Error code
What happened?
Possible error
Procedure
Err 8
Device possibly generated an arc
discharge leading to incomplete
program execution. A substantial
reduction in efficiency and
viability must be assumed.
Inappropriate cuvette,
Nucleofector® Solution or volume
in the cuvette. Device possibly
defective.
Clear the error message by
pressing any button. Check the
volume of Nucleofector® Solution.
Try again with a new cuvette.
Err 11
No pulse generated.
The device case is open.
Switch the device off, check
the correct mounting of all side
panels and the interlock between
all modules. Try again with the
same samples.
Err 12
Pulse possibly omitted.
Internal device temperature too
high for pulsing.
Switch off the device, wait for
2 minutes and run experiment
with a new cuvette.
Err 13
No pulse generated.
The device flap is open.
Check proper closing of flap after
the tray is closed and rerun the
experiment.
21
5.0Purchaser Notification
Purchaser Notification
The purchase of the 4D-Nucleofector® conveys to the buyer
a non-transferable, non-exclusive license without the right to
grant sublicenses limited for the use of Lonza’s proprietary
Nucleofector® process and method.
The license is limited to research use only. The license does not
include the right to use the purchased device, its components
or materials for any Commercial Purpose as defined below.
Notwithstanding the foregoing, the buyer shall be allowed to use
the licensed process in combination with the purchased device to
perform services internally for affiliates, subsidiaries, sections,
departments or scientific collaborators, provided that such
services are not for any Commercial Purpose.
Under no circumstances shall the licensed process and/or the
purchased device be used for testing or treatment in humans.
The buyer acknowledges that the licensed process and/or the
purchased device are not diagnostic or therapeutic tools and that
they shall not be used as an accessory or complement to such
tools.
Commercial Purposes as used herein means any activity done
by a party for monetary or other consideration and may include,
but is not limited to: (1) use of the product or its components
in manufacturing; (2) use of the product or its components to
provide a service, information, or data; (3) use of the product or its
components for therapeutic, diagnostic or prophylactic purposes;
or (4) resale of the product or its components, whether or not such
product or its components are resold for use in research.
The buyer is not licensed to manufacture, copy, reproduce,
transmit, distribute, sell, lease, transfer or sublicense the licensed
process and/or the purchased device.
For information on purchasing a license to this product for purposes other than research, contact our Licensing Department, at
+49 221 99199 0 or e-mail: [email protected]
Representations and Warranties
Lonza represents and warrants that, it is not aware of any legal
deficiencies of the Patents and that to its knowledge it has the
right to dispose of the patents. Lonza further represents and
warrants that it has no knowledge of any rights of third parties
which have priority over the patents and that the inventors of the
invention falling under the patents have transferred all rights and
entitlements to Lonza and that accordingly Lonza has full power
and authority to grant the license provided hereunder.
Lonza does not warrant that the information and data pertaining
to the licensed process and/or the purchased device is correct
and without defects, that the use of such information or data is
adequate for the use of the licensed process and/or the purchased
device or that the technical information or data for the licensed
process and/or the purchased device is complete.
Lonza does not warrant either that the use of the license does not
infringe third parties’ rights or does not cause damages to third
parties. Any liability for later invalidation or lapse of the patents
is excluded.
Purchaser Notification 5.0
22
Subject to the warranty granted for the Purchased Device, all
warranty claims are excluded as far as they are based on the
Licensed Process or the Purchased Device. No warranty is made
for the reliability, the quality, the economic utilization, efficiency,
merchantability, fitness for purpose and the usefulness of the
Licensed Process, the Purchased Device, or products resulting
from the use of the licensed process or the purchased device, for
the intended purpose or for any other purpose. The exclusion of
warranty claims does not apply in case of gross negligence or
intentional misconduct of Lonza.
Lonza does not assume any liability that the licensed process,
the Purchased Device or any product resulting from the use of the
licensed product shall be approved by governmental authorities or
any other institution or supervisory board or similar agencies.
23
6.0Appendix
Technical data
Power supply
100 – 110 VAC or 230 VAC
50 – 60 Hz
self-regulating
Power consumption
140 VA
Fuse protection
Fuse T2.5A, L250V
Temperature range
+15°C to +40°C, non-condensing
Safety class
EN 61010-1
UL 61010A-1
IP 20
Weight
8.0 kg (17.8 lb); system comprising Core Unit and X Unit
Dimensions
(w x h x d)
45 x 10.5 x 28 cm; system comprising Core Unit and X Unit
(17.7 x 4.1 x 11.0 in)
Manufacturing date
The manufacturing year is encoded by the second and third digit of the serial number, e.g., serial number x10xxxxx
was manufactured in 2010.
Firmware update
https://www.lonzabio.com/cell-biology/transfection/technology/nucleofector/nucleofector-program/
Appendix 6.0
24
Notes
25
Notes
Notes
Contact Information
North America
Customer Service:
Scientific Support:
[email protected]
Online Ordering:
800 638 8174 (toll free)
800 521 0390 (toll free)
www.lonza.com
Europe
Customer Service:
+ 32 87 321 611
Research Solutions
Scientific Support:
+ 49 221 99199 400
[email protected]
Research Solutions,
Therapeutic Cell Solutions, Testing Solutions
Scientific Support:
+ 32 87 321 611
[email protected]
Online Ordering:
www.lonza.com
International
Contact your local Lonza Distributor
Customer Service:
+ 1 301 898 7025, ext. 2400
[email protected]
International Offices
Australia
Austria
Belgium
Brazil
Denmark
France
Germany
India
Ireland
Italy
Japan
Poland
Singapore
Spain
Sweden
Switzerland
The Netherlands
United Kingdom
+ 61 3 9550 0883
0800 201 538 (toll free)
+ 32 87 321 611
+ 55 11 2069 8800
+ 45 43 56 74 00
0800 91 19 81 (toll free)
0800 182 52 87 (toll free)
+ 91 22 4342 4000
1800654253 (toll free)
+ 39 0363 45710
+ 81 3 5566 0612
+ 48 781 120 300
+ 65 6521 4379
+ 34 902 531 366
020 140 4410 (toll free)
0800 83 86 20 (toll free)
0800 022 4525 (toll free)
0808 234 97 88 (toll free)
Lonza Cologne AG
50829 Cologne, Germany
For Research Use Only. Not for use in diagnostic procedures.
Manufacturer and distributor information: The 4D-Nucleofector® System is manufactured by Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany and
distributed in the US by Lonza Walkersville, Inc. (8830 Biggs Ford Road, Walkersville,
MD 21793).
The use of this product, alone or in combination with materials and/or methods of
others, may require a license from a third party. User shall be fully responsible for
determining whether and from whom it requires such license and for obtaining such
license.
The Amaxa® Nucleofector® Technology is covered by patent and/or patent pending
rights owned by Lonza Cologne AG.
Other product and company names mentioned herein are the trademarks of their
respective owners.
The information contained herein is believed to be correct and corresponds to the
latest state of scientific and technical knowledge. However, no warranty is made,
either expressed or implied, regarding its accuracy or the results to be obtained
from the use of such information and no warranty is expressed or implied concerning
the use of these products. The buyer assumes all risks of use and/or handling. No
statement is intended or should be construed as a recommendation to infringe any
existing patent.
© Copyright 2010, Lonza Cologne AG. All rights reserved.
TS-4D-Nucleo Manual-1 04/2010
CD-MN025