4D Nucleofector Manual
4D Nucleofector Manual
Support Teams
Europe
Scientific Support: +32 87 321 611
scientific.support.eu@lonza.com
North America
Scientific Support: +1 800 521 0390 (toll free)
scientific.support@lonza.com
The 4D-Nucleofector™ System 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, L fuses to replace blown fuses.
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. Furthermore, a system may consist
of minor model variants of the units. These variants can be discriminated
by their part codes and serial numbers (table 2.1).
A typical 4D-Nucleofector™ System includes: Figure 2.2: 4D-Nucleofector™ System comprising Core, X and Y Unit (A) or Core, X and
1. One 4D-Nucleofector™ Core Unit LV Unit (B)
2. At least one functional unit
– X Unit: comprising two retainers for 100 μL Nucleocuvette™ Vessels A B
and one retainer for 16-well Nucleocuvette™ Strips
– Y Unit: enabling transfection of cells in adherence using a 24-well
5
Dipping Electrode Array
– LV Unit: for large volume transfection of up to 109 cells
3. Power cord
6
4. Interface cable for high voltage and data connection (plus termination
7
plug cap, not shown)
8
10
C
B
E D
This section gives an overview of the 4D-Nucleofector™ System operating The touch screen of the 4D-Nucleofector™ System can be set at four
software. Details of the Nucleofection process can be found in the cell- angles (0°, 30°, 45°, and 60°), providing convenient access for the user.
type specific Optimized Protocols (www.lonza.com/optimized-protocols). Press the label 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
NOTE: All screenshots shown in this section refer to software version most convenient angle by simply pulling it into a more upright position. The
4.0. screen can be returned to its home position by moving it to the 60° position
and then pulling it gently forward towards the front of the Core Unit. A
2.7.1 Turning on the 4D-Nucleofector™ System 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.
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 NOTE:
Do not try to close the display when the system is switched
moments — and the blue LED at the front of the Core Unit will be lit. off. If there is a need to close it during OFF status or in case the
Once the start-up procedure is complete, the 4D-Nucleofector™ graphical display gets stuck, use the release knob shown in figure 2.7
user interface (touch screen) will display the software main screen. (red circle).
The main screen (figure 2.6) displays the current configuration of the
4D-Nucleofector™ System (Core Unit and functional units). Figure 2.7: Releasing the touch screen
1 2
Figure 2.6: Main screen
3 4
5 6
Emergency release
Lock icon (4) Activates transport lock (for X and Y Unit) and shuts down the
system (software version 4.0 or later, in older versions it may not
be available or only activate the transport mode)
BACK Return to previous screen
CLR Reset settings for an experiment or a Nucleofection vessel
SAVE Save experiment or results file
LOAD Load predefined experiments
OK/START Confirm selection or execute a program
A-Z, Z-A, Top 10, Last Sort a list alphabetically, display the most frequent 10 items or the
10 most recent 10 items
Magnifier symbol Activate search functions
Figure 2.9: System shut down i Display additional information (text)
<, > Arrow keys to switch between experimental setup screens
A B
This chapter explains how to turn off the 4D-Nucleofector™ System properly.
Following this procedure will guarantee a safe shut down of the system
and protection against damage of interior fragile parts. The described
procedure is valid for software version 4.0 or newer. Older versions may
not provide a software shut down (version 2.12 - 2.15) or only offer the
transport mode (version 2.16). It is strongly recommended to upgrade
the system to the newest software version (chapter 2.12.7).
–– Press the “Lock” icon (figure 2.9, A, circle). Alternatively you can
press the “Wrench” icon and select “Shut Down”.
–– A message will appear asking whether you want to shut down the
system (figure 2.9, B). Upon pressing “YES”, first the transport lock
C D will be applied (for X or Y Unit) (figure 2.9, C).
–– Wait until you receive the message “You can now switch off the system”
NOTE: By pressing “BACK” you would release the transport lock again.
–– Finally, switch off the device via the main power switch located at
the rear of the Core Unit. If the system was turned off via the power
switch directly (without previous software shut down), a reminder will
appear once you turn the system on again next time (figure 2.9, D).
2.8.1 Overview
Use As Is Modify
Load Samples
(chapter 2.9, 2.10 or 2.11)
A 1. Select the functional unit (X, Y or LV) you wish to work with by pressing
the appropriate icon (figure 2.10, A).
2. For X and LV Unit: A screen appears displaying the Nucleofection
vessels available for the selected functional unit (figure 2.10, B, C).
Press the icon displaying the vessel type you wish to use.
B C
Parameter Description
Cell Type Program Using the “Cell Type Program” option is the easiest way to select parameters for a vessel or well or group of those.
Each “Cell Type Program” is assigned to the optimal program for the certain cell type as defined in the Optimized Protocol
(e.g., “T cells, human, unstim.” Is affiliated to the optimal Nucleofector™ Program and Solution for unstimulated human T
cells).
The “Cell Type Program” list can be extended by the user with customized cell-type codes (e.g., after a cell line optimization;
2.12.3). Lonza defined programs are highlighted in blue while custom program codes will be highlighted in black.
Solution In case the recommended Nucleofector™ Solution is not selected automatically via the “Cell Type Program” option, the
“Solution” can be selected manually.
Pulse Code In case the recommended Nucleofector™ Program is not selected automatically via the “Cell Type Program” option, a “Pulse
Code” (e.g. FI-115) can be entered manually.
Note: For manual “Pulse Code” selection, the “Solution” has to be selected first (see below).
Control 1. Sample: This is the default setting and defines a normal sample containing cells and substrate. The selected
(X and Y Unit) Nucleofection program will be applied to this position.
2. No DNA: Negative control. Nucleofection program applied to vessel with cells but without substrate
3. No program: Negative control. No Nucleofection program applied to a vessel containing cells and substrate
In case of “Sample” or “No DNA” the selection has only an informative purpose. The selection of “No program” has a
functional effect, i.e. no pulse will be applied.
Volume (LV Unit with When using the scalable cuvette version, the LV Nucleocuvette™ Cartridge, the volume that should be processed has to be
LV Nucleocuvette™ defined. Depending on the number of reservoirs used, one or two volume parameters must be specified. The maximum total
Cartridge) volume that can be entered is 20 mL.
1. After unit and vessel type selection (see 2.8.2) a screen appears with
a field showing “Choose Experiment or Position” (figure 2.11, A).
2. Press on the touch field labeled “Choose Experiment”
3. A list of predefined experiments will appear (figure 2.11, B). The
list comprises experiments pre-defined by Lonza (template files;
blue) and all experiments saved by the user (black).
4. Select the desired experiment by tapping on the appropriate line of
the list. The experiment selected will be highlighted. Confirm your C D
selection by pressing “OK”.
5. By pressing on the icons displaying the Nucleofection vessels or
wells you can check the settings (“Cell Type Program,” “Pulse Code,”
etc.) for the selected well (figure 2.11, C).
6. In case you want to modify the settings of a predefined experiment,
click on the well(s) you want to change and adapt the settings (for
unit-specific details see chapters 2.9, 2.10 or 2.11). An asterisk (*)
will be added to the experiment file name to indicate the deviation
from the original file. You can save the changed experiments under
a new name by pressing “SAVE”. Alternatively you may review the
summary first (see next step) and then save it.
7. To accept all settings, press “OK”.
8. A summary of the experimental settings will appear (figure 2.11, D).
If the experimental setup is as desired, load the samples (for unit-
specific details see chapters 2.9.1, 2.10 or 2.11).
2
2.9.1 Defining a New Experiment
Figure 2.13: Experiment definition (X Unit; example: 16-well
Nucleocuvette™ Strips) 1. After unit and vessel type selection (see chapter 2.8.2) a screen
appears with the message “Choose Experiment or Position” (figure
A B
2.13, A). You can now either select a predefined experiment (see
chapter 2.8.4) or define an experiment from the beginning.
By tapping on a position you can select one or multiple samples/wells
(e.g. A1 and A2) that should be defined with the same parameters.
The selected positions are highlighted with an orange frame (figure
2.13, B).
NOTE: For the 16-well Nucleocuvette™ Strips, you can select a whole
column by double clicking on the top or bottom well. Wells can
be de-selected by tapping on the position again. The orange frame
will disappear.
2. Upon well selection the fields “CELL TYPE PROGRAM”, “CUSTOM
C D
PROGRAM”, “PULSE CODE”, “SOLUTION” and “CONTROL” will be activated
(figure 2.13, B). For further explanation about parameters, please refer
to chapter 2.8.3.
3. Press the field “CELL TYPE PROGRAM” to choose predefined
Nucleofection conditions from a cell type list (figure 2.13, C). Use the
search (magnifying glass symbol) or the sort list functions (A-Z) to
find conditions more quickly.
a. Select the desired cell type code 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”.
b. To confirm your selection press “OK”.
c. If required, modify pulse code by pressing the letter or number
E F code fields. A keyboard will appear, enabling you to change
settings. The solution code can be modified via a selection list.
NOTE: Instead of defining solution and program code via the CELL TYPE
PROGRAM, both parameters can also be selected manually, e.g.
in case no predefined cell type program is available. For adding
new cell type programs, please refer to chapter 2.7.4.
4. Define control options for the selected vessel by choosing
(figure 2.13, D)
5. Optional: At this stage (or at step 7) you can save your defined
experiment for future use by pressing the “SAVE” button. 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 (figure 2.13, E).
6. Confirm and save the experiment parameters by pressing “OK” (saves
the experiment and opens the drawer) or “SAVE” (saves the experiment
for later use).
7. A summary of the defined settings will be displayed (figure 2.13, F).
Please check for correctness before loading samples (see chapter
2.9.2) and starting the experiments by pressing “START” (see chapter
2.9.3)
E F
G H
A B 10. After loading the samples press “START” to run the experiment (figure
2.15, A).
11. The progress of the experiment is indicated by changing the color
of the cuvette or well positions (figure 2.15, B) (for color codes see
chapter 4).
NOTE: When working with the single 100 µL Nucleocuvette™ Vessels you
can process two cuvettes at once. If more than two cuvettes have
been defined the drawer opens automatically after each set of
two and a message “Please change cuvette(s). Proceed?” will be 2
displayed (figure 2.15, C). Load next samples and press “YES” to
continue or press “NO” to interrupt the experiment.
12. When the experiment is complete, a result file summarizing the
C D Nucleofection process will be displayed (figure 2.15, B).
13. The result file will be saved automatically by the system and can be
reopened as described in chapter 2.12.2.
14. You can repeat the same experiment by pressing “START” again.
A message will appear asking “Do you want to pulse the same
experiment again?” (figure 2.15, D).
15. Press “OK” to start the experiment, “NEW” to return to the “Experiment”
screen and define a new experiment or “CANCEL” to return to the
“Results” screen.
1. After unit selection (see chapter 2.8.2) a screen appears that allows
you to define a new experiment or select a previously saved experiment
by pressing “Choose existing experiment” (figure 2.16, A).
2. When defining a new experiment, by tapping on a position you can
select one or multiple samples/wells (e.g. A1 and A2) that should
be defined with the same parameters. The selected positions are
highlighted with an orange frame (figure 2.16, B).
NOTE: You can select a whole column by double clicking on the top or
bottom well. Wells can be de-selected by tapping on the position
again. The orange frame will disappear.
C D
3. Upon well selection the fields “CELL TYPE PROGRAM”, “CUSTOM
PROGRAM”, “PULSE CODE”, “SOLUTION” and “CONTROL” will be activated
(2). For further explanation about parameters, please refer to chapter
2.8.3.
4. Press the field “CELL TYPE PROGRAM” to choose predefined
Nucleofection conditions from a cell type list (figure 2.16, C). Use the
search (magnifying glass symbol) or the sort list functions (A-Z) to
find conditions more quickly.
a. Select the desired cell type code 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”.
b. To confirm your selection press “OK”. E F
c. If required, modify pulse code by pressing the letter or number
code fields. A keyboard will appear, enabling you to change
settings. The solution code can be modified via a selection list.
NOTE: Instead of defining solution and program code via the CELL TYPE
PROGRAM, both parameters can also be selected manually, e.g.
in case no predefined cell type program is available. For adding
new cell type programs, please refer to chapter 2.12.3.
5. Define control options for the selected well by choosing
(figure 2.16, E)
6. Optional: At this stage (or at step 8) you can save your defined
experiment for future use by pressing the “SAVE” button. A keyboard
will appear allowing you to define a name (max. length: 26 characters). G
You may enter further information about your experiment by touching
the “Info” field and typing in your text (figure 2.16, F).
7. Confirm and save the experiment parameters by pressing “OK” (saves
the experiment and opens the drawer) or “SAVE” (saves the experiment
for later use).
8. A summary of the defined settings will be displayed (figure 2.16, G).
Please check for correctness before loading samples (see chapter
2.10.2) and starting the experiments by pressing “START” (see chapter
2.10.3).
9. Insert the 24-well Dipping Electrode Array into the 24-well culture plate
containing your Nucleofection samples. Make sure that the dipping
electrode array is inserted in the right orientation.
10. Place 24-well plate with inserted dipping electrode array into the
retainer of the 4D-Nucleofector™ Y Unit. Well “A1” must be in upper
left position. If the array-plate sandwich was entered in the wrong
orientation an error message will appear after pressing “START”.
11. It is not recommended to re-use dipping electrodes as this may lead
to suboptimal transfection efficiencies. An RFID chip contained in 2
the dipping electrode lid logs usages and after pressing “START” the
software will check for used wells and offer you different options how
to continue.
A
The 4D-Nucleofector™ LV Unit can handle two types of vessels, the fixed
volume 1 mL Nucleocuvette™ Cartridge for cell numbers up to 1x108 cells
and the scalable LV Nucleocuvette™ Cartridge for cell numbers up to 1x109
cells. 4
2. Contacting side/part/area
3. Injection port (rear side)
4. Air outlet port with 0.2 µM sterile filter
3
A B
11. When the cartridge is inserted, press “START” to initiate the
Nucleofection process (figure 2.22, A).
12. The progress of the experiment is indicated by a progress bar (figure
2.22, B).
13. When the experiment is complete, a result file summarizing the
Nucleofection process will be displayed.
14. The result file will be saved automatically by the system and can
be reopened as described in chapter 2.12.2. The overall result is
indicated by a color code: green for “OK” (figure 2.22, C), yellow in
case of few errors and red in case of multiple errors (figure 2.22, D).
If errors occurred you can check for more details by pressing “More
details”. The result details (figure 2.22, E) will display the type of error D
C
and its percent occurrence in the processed volume. For additional
information on the error press “i”.
15. Once the process is completed take out the cartridge by pressing the
black levers.
16. Aspirate the transfected cell suspension and transfer cells into culture.
Optionally you may wash the cartridge with medium to aspirate
residual liquid.
17. You can repeat the same experiment by pressing “START” again.
8a
2
7
3
5
6 1
8b 8c
1. Cartridge inserted in slot 7. Holder for cell suspension reservoir placed on a magnetic
2. Short venting tube with filter attached to the male Luer connector on the front plate stirring platform (stirring speed to approx. 300 rpm)
3. Tubes inserted in pinch valves (press black round surface of the valves while pulling 8. All reservoir-tube connections established to
tubes fully into the valves) a. Input reservoir for cell suspension
4. T connector inserted into T connector holder. Two larger diameter tubes completely b. Input reservoir for substrate (optional)
inserted into liquid sensors c. Output reservoir
5. Upper tube (for cell suspension!) inserted into the upper pump. Flap closed.
6. Optional - when feeding substrate separately - lower tube inserted into lower pump.
Flap closed.
E F
G H
This chapter explains how to change settings like date and time and how
to organize and manage your stored experiments, results and custom
programs.
–– Activate the “Settings” menu by either pressing the “Core Unit” icon or
the “Wrench” icon submenu “Settings” (figure 2.27, A).
–– A screen appears displaying a list of setting options (figure 2.27, B):
–– Experiments – display user-defined experiments (see chapter
2.12.1 for more information)
–– Custom programs – create and manage custom Nucleofection
programs (see chapter 2.12.3 for more information)
–– Results – reopen or load result files onto a USB stick plugged
into the USB port in front of the Core Unit (see chapter 2.12.2 for
more information)
–– Lonza programs – version information about the cell type list and
option to upload a new cell type program list (see chapter 2.12.6)
–– 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 keyboard – change language settings for display
functions and keyboard
–– ... more – to switch between pages of the settings list
–– Firmware update – update the system software (see chapter
2.12.7for details)
–– Device cleaning – opens the tray of the functional modules to
remove it for cleaning (see chapter 2.12.5 for details)
–– System restart – restart system
–– Shut down – activates the transportation lock (for X and Y Unit)
and shuts down the system (software version 4.0 or later, in older
versions it may not be available or only activate the transport
mode; also see chapter 2.7.4)
–– Synchronize – supports synchronization of the actual firmware
with the PC Editor (see chapter 2.12.4 and chapter 2.13 for more
information)
–– Version – displays firmware version and serial number of all
modules
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 described below.
2.12.1 Experiments
C Menu “Experiments” D Menu “Results”
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 (figure 2.27, C).
G Menu “ Synchronization” H Menu “ Synchronization” The software offers the possibility to add custom program codes for
choosing a defined combination of a Nucleofector™ Program and Solution,
e.g., after an optimization. Custom program codes are included in
alphabetical order into the “Cell Type Program” list and can be differentiated
from Lonza defined code by color:
Blue: Lonza defined cell type program codes
Black: Customer defined codes. 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” (figure 2.27, E).
This menu is used to synchronize the firmware of your 4D-Nucleofector™ The “Cell Type Program” list can be exchanged independently from a
System with the PC Editor software (see chapter 2.13). It is necessary to firmware update (see chapter 2.12.7). For uploading a new “Cell Type
perform synchronization with each firmware update. Program” list follow instructions below:
–– Open the “Settings” menu.
To start synchronization: –– Press “LONZA Programs” (figure 2.27, K).
–– Insert a USB stick into the USB port on the front of the Core Unit. –– Insert a USB stick comprising the file you want to upload (*.pd)
–– Press “Synchronize” (figure 2.17, G). –– Press “Load” and browse for file location.
–– The 4D-Nucleofector™ System is now creating a synchronization file –– Double-click on USB folder and select new file.
and saves it to the USB stick (figure 2.27, H). –– Press “OK” for upload.
–– Remove the USB stick and connect it to the PC your PC Editor software
is installed. 2.12.7 Firmware Update
–– Open the PC Editor software as described in chapter 2.13
–– Select the menu Settings and press “Synchronize”. Due to technical improvement of the operation software, the
–– A window will open enabling you to select the USB stick. 4D-Nucleofector™ System may require an update of its firmware. To update
–– Select the USB stick as storage device and press “OK”. the firmware proceed as follows:
–– Confirm that you’d like to update the PC Editor by pressing “YES” or –– Start the 4D-Nucleofector™ System.
cancel by pressing “NO”. –– The “Choose a Device” main screen will appear.
–– Click on the Core Unit icon.
2.12.5 Cleaning the Nucleofection –– Select “Firmware Update” within the “Settings” menu.
Vessel Tray (X or Y Unit) –– A dialog box will appear asking to plug in the USB stick.
–– Plug in the USB stick containing the new firmware (as provided by
To allow convenient cleaning, the Nucleofection vessel tray can be Lonza) and press UPD” (figure 2.27, L).
removed from the X and the Y Unit. To dismount the tray please follow –– Follow the instructions on the screen to update the firmware.
the instructions below: NOTE: More comprehensive firmware updates may not be possible via
–– Open the “Settings” menu USB upload and require return of the system to Lonza.
–– 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.
D E
3 Troubleshooting
3.1 Suboptimal Transfection Results
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 our Scientific Support Team.
A B
This chapter describes how to rescue your samples from the X or Y Unit
in case of system malfunction or break down of electricity. Depending
on the model variant (see table 2.1 on page 8) the process differs
slightly (figure 3.1).
C D
E F
Parameter File Result File, OK Result File, Warning Result File, Error Skipped Well
Undefined
Sample
No Program
No DNA
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 informa-
tion 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 Lonza’s intellectual property is excluded.
Core Unit
X or Y Unit
LV Unit
Europe
Customer Service: +32 87 321 611
order.europe@lonza.com
Scientific Support: +32 87 321 611
scientific.support.eu@lonza.com
International
Contact your local Lonza distributor
Customer Service: +1 301 898 7025
Fax: +1 301 845 8291
scientific.support@lonza.com
International Offices
Australia + 61 3 9550 0883
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