Lonza 4D-Nucleofector User manual

Your guide to setup and usage of the
4D-Nucleofector® System
For research use only
4D-Nucleofector®
Hardware
Manual

2 | 4D-Nucleofector® Hardware Manual
The purchase of the 4D-Nucleofector® System conveys to
the buyer the non-transferable right to use the system as
well as Lonza’s proprietary Nucleofector® 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.

4D-Nucleofector® Hardware Manual | 3
Content
1 Nucleofector®Technology 4
How it works 4
Transfection of any cell 4
Transfection of any substrate 4
Moving transfection into the fourth dimension 5
Experience the advanced Nucleofector® Platform: 5
2 Operating instructions 6
2.1 Restrictions 6
2.2 Maintenance 6
2.3 Safety instructions – please read carefully 6
2.3.1 Consignes de sécurité - à lire attentivement 7
2.4 Waste disposal 8
2.5 4D-Nucleofector® System components 9
2.6 Set-up instructions 10
2.7 General use instructions 10
2.7.1 Turning on the 4D-Nucleofector® System 10
2.7.2 Operating software 10
2.7.3 Turning off the 4D-Nucleofector® System 11
2.8 General instructions for running a
Nucleofection® Experiment 11
2.8.1 Overview 11
2.8.2 Unit and vessel selection 11
2.8.3 Experiment parameters – overview 12
2.8.4 Using a predefined experiment 13
2.9 4D-Nucleofector® X Unit 14
2.9.1 Defining a new experiment 14
2.9.2 Loading samples 16
2.9.3 Running the experiment 16
2.10 4D-Nucleofector® Y Unit 17
2.10.1 Defining a new experiment 17
2.10.2 Loading samples 18
2.10.3 Running the experiment 18
2.11 4D-Nucleofector® 96-well Unit 19
2.11.1 Defining a new experiment 19
2.11.2 Loading samples 21
2.11.3 Running the experiment 21
2.12 4D-Nucleofector® LV Unit 22
2.12.1 Using the 1 mL Nucleocuvette® Cartridge (Fixed volume) 23
2.12.1.1 Defining a new experiment 23
2.12.1.2 Loading Samples 24
2.12.1.3 Running the experiment 24
2.12.2 Using the LV Nucleocuvette® Cartridge (Scalable volume) 25
2.12.2.1 Defining a new experiment 26
2.12.2.2 Mounting an LV Nucleocuvette® Cartridge 26
2.12.2.3 Loading Samples 28
2.12.2.4 Running the experiment 30
2.13 Additional settings and options 31
2.13.1 Settings menu on the right 31
2.13.1.1 System settings 31
2.13.1.2 Cleaning the Nucleofection® Vessel Tray (X, Y, or 96-well Unit) 31
2.13.2 Left menu 32
2.13.2.1 E xperiments 32
2.13.2.2 Results 32
2.13.2.3 Custom Cell Type Codes 32
2.13.2 Lonza programs 33
2.13.4 Firmware update 33
3 Troubleshooting 34
3.1 Suboptimal transfection results 34
3.2 Rescue of samples (X and Y Unit) 35
4 Error codes of the
4D-Nucleofector®System 36
4.1 Color coding of wells 36
4.2 Error codes 36
4.3 Arc discharge correction 37
5 Purchaser notication 37
Purchaser notification 37
Representations and warranties 37
6 Technical specications 39

4 | 4D-Nucleofector® Hardware Manual
1 Nucleofector®Technology
Since its introduction in 2001, Nucleofector® Technology has
transformed transfection. Nucleofection® Technology 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 reect how Nucleofector® Technology is
driving research in numerous cell types and applications. Nucleofector®
Technology provides an answer to almost every transfection challenge.
How it works
Nucleofector® Technology is based on two unique components,
the Nucleofector® System that delivers the specically optimized
electrical parameters and Nucleofector® Kits, which contain specic
Nucleofector® Solutions. As part of Nucleofector® Technology, Lonza
provides cell-type specic optimized protocols for many dierent cell
lines and primary cells.
Transfection of any cell
With over 325 protocols optimized by Lonza’s R&D Team for cell
lines and primary cells and more than 750 entries in the online Cell
Transfection Database, Nucleofector® Technology is the transfection
method of choice for dicult-to-transfect cell types. Moreover,
the Nucleofection® Method is the only electroporation-based
technology allowing reliable transfection of adherent cells and cells
in suspension.
Transfection of any substrate
Nucleofector® Technology oers high exibility 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® Technology an ideal tool for providing answers
to scientic questions in over-expression studies, gene silencing
approaches, protein expression, generation of stable clones and
many more applications besides.
www.lonza.com/celldatabase

4D-Nucleofector® Hardware Manual | 5
The 4D-Nucleofector® System
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 oer advanced performance, exibility 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 advanced Nucleofector® Platform:
• A novel conductive polymer based cuvette system completely
free of toxic metal ions
• Transfection of dierent cell numbers using the same conditions
• Nucleofection® Conditions for cells in adherence
• Closed, scalable transfection of larger cell numbers in the range of
1x107to 1x109cells per reaction
lonza.com/transfection
Scientic Support Teams
Europe
Phone: [phone]
E-mail: scienti[e-mail]
North America
Phone: [phone] (toll free)
E-mail: scienti[e-mail]
1

6 | 4D-Nucleofector® Hardware Manual
2.1 Restrictions
Medical use restrictions
Nucleofector® Technology is intended for research and investigational
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 scientic 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 and development purposes, the terms of which are disclosed
in detail in chapter 5. Certain commercial application is allowed under
Lonza’s license for for-prot-entities, as set forth in the license.
For license information contact Lonza Cologne GmbH:
Phone: [phone]
E-mail: [e-mail]
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. Do not use any aerosols for cleaning. 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® System. However, the system should
be removed from the hood during extended UV irradiation.
The 4D-Nucleofector® System is protected by two main fuses. Both are
inside a receptacle incorporated in the power socket of the Core Unit
(see gure 2.1). Blown fuses can easily be replaced. Disconnect the
4D-Nucleofector® System from the power supply and insert a small
at 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.
Figure 2.1: Opened fuse receptacles
The 4D-Nucleofector® System will not function unless both the left
and right receptacle contain intact fuses. Blown fuses can usually be
identied by molten broken wires inside the glass tube.
Only use T2.5A, L fuses to replace blown fuses.
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 certied 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 cells.
These electrical impulses can be deadly!
2 Operating instructions

4D-Nucleofector® Hardware Manual | 7
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 user-
serviceable 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
Nucleofection® 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® System. They are designed to connect certified
add-on systems to the 4D-Nucleofector® System.
• Only use the system when it is set on top of a safe, leveled 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 explosion-endangered
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 Nucleofection® Vessels (100 µL Nucleocuvette®
Vessel or 20 µL Nucleocuvette® Strip for X Unit; 1 mL or LV
Nucleocuvette® Cartridge for LV Unit; 24-well Dipping Electrode Array
for Y Unit). Using consumables from any other source than Lonza
will preclude all warranty and liability claims.
• Do not alter the setup of the pre-assembled LV Nucleocuvette®
Cartridge.
• Nucleofector® Solutions used with the Nucleofector® I/II/2b System
are not compatible with the 4D-Nucleofector®System. Only use
dedicated 4D-Nucleofector® or 96-well Nucleofector® Solutions.
• Never unplug high voltage cable while system is running it will
destroy the device.
• Unpack the Nucleocuvette® Vessels or 24-well Dipping Electrode
Arrays 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.
To ensure that the system is safe to use, 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. To ensure that the system is safe to use,
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 cannot 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 leak-
proof 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.
2.3.1 Consignes de sécurité - à lire attentivement
Ce symbole signie qu‘il existe un risque de choc électrique. Un choc
électrique peut provoquer des blessures graves, voire mortelles.
L’appareil 4D-Nucleofector® a été certié par les normes de
sécurité internationales et peut être utilisé en toute sécurité
lorsqu’il est utilisé conformément à ce manuel.
• Cet appareil est conçu pour délivrer des impulsions électriques
variables à haute tension dans le but d‘introduire des substrats dans
des cellules eucaryotes.
Ces impulsions électriques peuvent être mortelles!
Par conséquent, utilisez cet appareil avec attention et prenez les
précautions suivantes:
• Utilisez l‘appareil uniquement après avoir lu et compris le manuel
4D-Nucleofector®. Le manuel devrait être accessible à tous les
utilisateurs. Assurez-vous que chaque utilisateur potentiel le lit et le
comprend.
• Ne pas ouvrir l‘appareil. L‘appareil ne contient pas de pièces
réparables par l‘utilisateur. Les composants du circuit ne doivent
en aucun cas être perturbés, car ils peuvent provoquer un choc
électrique même lorsque le système n‘est pas en fonctionnement.
• Ne modifiez pas l‘appareil de quelque manière que ce soit.
• Ne gênez pas le mouvement du tiroir maintenant les cuvettes de
Nucleofection® ou les plaques de cultures.
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8 | 4D-Nucleofector® Hardware Manual
• Ne branchez aucun connecteur non certifié par Lonza sur les prises
d‘électrodes haute-tension externes situées à l‘arrière du système
4D-Nucleofector®. Ils sont conçus pour connecter des systèmes
certifiés complémentaires au système 4D-Nucleofector®, par
exemple l’appareil X Unit ou 96-well Unit.
• Utilisez l‘appareil uniquement lorsqu‘il est installé sur une table ou
paillasse sûre, plate et stable.
• Placez l‘appareil de manière à pouvoir retirer facilement le cordon
d’alimentation à tout moment.
• N‘exposez pas l‘appareil à un environnement humide.
• L‘appareil ne doit pas être exposé à la lumière directe du soleil ni
être placé dans un environnement chaud
• L‘appareil n‘est pas approuvé pour une utilisation dans des zones
présentant un danger d‘incendie ou d‘explosion, ni pour une
utilisation avec des milieux inflammables ou explosifs.
• Prendre des précautions contre les chocs et les vibrations lors des
déplacements et transports de l’appareil 4D-Nucleofector®.
• Utilisez l’appareil avec les solutions et cuvettes Nucleofector®
certifiées par Lonza uniquement (cuvette Nucleocuvette® de 100
μL ou barrette Nucleocuvette® de 20 μL pour Unité X; cartouche
Nucleocuvette® de 1 mL ou LV pour Unité LV; plaque d‘électrodes à
immersion 24-puits pour Unité Y). L‘utilisation de consommable de
toute autre source que Lonza exclura toute réclamation appelant la
garantie et la responsabilité de Lonza.
• Ne modifiez pas la configuration de la cartouche Nucleocuvette® LV
préassemblée.
• Les solutions Nucleofector® pour Nucleofector®I/II/2b ne sont pas
compatibles avec le système 4D-Nucleofector®.
• Ne débranchez jamais le câble haute tension lorsque le système est
en marche.
• Déballez les Nucleocuvette® ou les plaques d‘électrodes à immersion
de 24-puits juste avant l‘expérience. Assurez-vous que les zones de
contact extérieures soient sèches.
• La sécurité peut être compromise si du liquide a été répandu à
proximité du système 4D-Nucleofector®. Assurez-vous qu‘aucun
fluide n‘est en contact avec ou n‘a pénétré dans le système.
• Si du liquide a pénétré l‘appareil, la sécurité peut être compromise.
Pour confirmer que l‘utilisation de l‘appareil est sécurisée, contactez
le Service du Support Scientifique de Lonza pour les actions ou les
précautions à prendre.
• Ne pas insérer ou placer d‘objets étrangers dans les tiroirs
électriques du système 4D-Nucleofector®.
• La sécurité peut être compromise si un objet étranger est entré dans
le système 4D-Nucleofector®. Pour vous assurer que le système est
sûr à utiliser, contactez le Service du Support Scientifique de Lonza
pour obtenir des conseils sur les actions et précautions à prendre.
• Si le système 4D-Nucleofector® a été endommagé, assurez-
vous qu‘il ne puisse être utilisé par aucun membre du personnel
et contactez le Service du Support Scientifique de Lonza pour
assistance.
• Toute réparation ne doit être effectuée que par un personnel autorisé
par Lonza.
• La manipulation des pièces de l‘appareil pouvant présenter
un risque de contamination des échantillons doit toujours être
effectuée avec des gants de protection. Toute élimination de ces
pièces doit être conforme aux procédures fédérales, régionales
ou nationales relatives à la manipulation et à l‘élimination des
déchets cliniques. Utilisez des conteneurs étanches et évitez toute
manipulation non protégée de telles pièces.
Lonza Cologne décline toute garantie et ne pourra en aucun cas être
tenue responsable de tout type de dommages causés par ou résultant
d‘une opération ou d‘une utilisation contraire aux consignes de sécurité
et de manipulation susmentionnées.
2.4 Waste disposal
Dispose of cuvettes, pipettes and Nucleofector® Solutions in a biohazard
container. Refer to your local waste management organization and to
the relevant laboratory safety instructions for proper disposal practices.

4D-Nucleofector® Hardware Manual | 9
2.5 4D-Nucleofector® System components
By denition the 4D-Nucleofector® System is a modular system oering
maximum exibility. 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).
Table 2.1: Overview of model variants
Model Variant A Variant B Variant C
Years 2010 – 2013 2013 – 2020 2021 - now
Part codes Core Unit: AAF-1001B
X Unit: AAF-1001X
Y Unit: AAF-1001Y
Core Unit: AAF-1002B
X Unit: AAF-1002X
Y Unit: AAF-1002Y
LV Unit: AAF-1002
Core Unit: AAF-1003B
X Unit: AAF-1003X
Y Unit: AAF-1003Y
LV Unit: AAF-1002LW
96-well: AAF-1009396
Serial
numbers
Core Unit: 5**B****
X Unit: 5**X****
Y Unit: 5**Y****
Core Unit: 8**B****
X Unit: 8**X****
Y Unit: 8**Y****
LV Unit: 9**LV***
Shuttle: 9**S****
Core Unit: 9**B2***
X Unit: 9**X2***
Y Unit: 9**Y2***
LV Unit: 9**LV2**
96-well: 9**S2***
Unit
arrangement
Horizontal or vertical
(via demountable
side panels)
Vertical only
(xed side panels)
Core Unit –
display frame
Silver Blue Blue
X/Y Unit – sample rescue using wheel accessible after removal of side panel via screw
behind termination plug (also see 3.2)
A typical 4D-Nucleofector® System includes (Figure 2.2 and
Figure 2.3 A and B):
1. 4D-Nucleofector® Core Unit
2. At least one functional unit
• X Unit: comprising two retainers for 100 μL Nucleocuvette®
Vessels and one retainer for 16-well Nucleocuvette® Strips
• Y Unit: enabling transfection of cells in adherence using a
24-well Dipping Electrode Array
• 96-well Unit: for medium throughput transfection of up to
96 samples in 20 µL format
• LV Unit: for large volume transfection of up to 109cells
Figure 2.2: 4D-Nucleofector® System in different configurations. Up to three functional
Nucleofector® Units can be operated with on Nucleofector® Core Unit.
1. Touch screen (graphical user interface of the system)
2. Power Switch LED
3. USB ports (for USB stick)
Figure 2.3: (A) 4D-Nucleofector® Core Unit, (B) 4D-Nucleofector® System comprising of
Core, X, and 96-well Unit in rear view.
4. Power cord socket with main power switch and fuses
5. Power cord
6. USB port(s)
7. Lonza interface outlet port to connect functional units
8. Lonza interface inlet port to connect functional units
9. Lonza interface inlet port of last functional unit
10. Lonza interface cable connected to outlet and inlet port
11. Lonza interface outlet port of functional unit
12. Lonza outlet port of last functional unit with termination plug
1
2
3
47
11
8
12
6
5
10
9
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10 | 4D-Nucleofector® Hardware Manual
2.6 Set-up instructions
1. Unpack the components of the 4D-Nucleofector® System and
check for completeness.
2. Stack the units with the Core Unit (containing the touch screen)
on top (Figure 2.3)
3. Connect the units (Figure 2.3) by using the interface cables:
• Connect the interface outlet port of the Core Unit (9) with the
interface inlet port of the rst functional unit (X Y, or 96-well
Unit; 10). Make sure that the cable is securely attached and
that the retaining screws on the cable housing are screwed
tightly into the port.
• Further functional units are added by connecting the outlet
(13) and the inlet port (11) of adjacent units as described
above.
• Important: Plug in the interface terminator cap into the outlet
of the last unit (14).
4. Attach the power cord (7) to the power cord socket (6) 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 rst
time.
The 4D-Nucleofector® System comes with default programs and
includes USB ports at the front and rear for software updates and
import and export of experiments and results.
2.7 General use instructions
This section gives an overview of the 4D-Nucleofector® System
operating software. Details of the Nucleofection® Process can
be found in the cell-type specic Optimized Protocols
(https://knowledge.lonza.com).
NOTE: All screenshots shown in this section refer to software
version 5.0.
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 blue LED at the front of the Core Unit will be lit. Then
press the power button at the front of the Core Unit. The system will
boot — this process may take a few moments. Once the start-up
procedure is complete, the 4D-Nucleofector® Graphical User Interface
(touch screen) will display the software main screen. The main
screen (home screen, gure 2.4) displays all vessel types that can
be used with the respective functional units that are attached to the
Core Unit. It is possible to connect up to three functional units with
one 4D-Nucleofector® Core Unit. In addition, at the bottom the recent
results and the top experiments used are shown.
2.7.2 Operating software
The 4D-Nucleofector® System is operated via an 8-inch touch screen
display and is controlled by intuitive software. Figure 2.4 and Table 2.2
will familiarize you with the functions of the touch fields and the basic
features of the operating software.
Figure 2.4: Main screen (home Screen) elements and general icons.
Table 2.2: Main menu and general commands or icons
Menu item Description
Accordion menu (1)Return to the main screen (home screen)
Experiment section – Search, import and export of
experiments
Results section – Search import and export of results
Cell type section – search for, create new and edit
existing cell type specic protocols
Time and date (2)Shows time and date (changes can be made in the
settings menu
Gear wheel icon (3)
Settings menus
Pick option for settings such as
• Date and date format (select date and dierent
formats to display)
• Time and time format (setting time and switching
between 24 h and am/pm display)
• Display brightness (Slide to adjust the brightness)
• Volume or Mute sound (Slide to adjust volume)
• Switching system to transportation mode
(transportation mode is applied each time the
system shuts down)
• Experiment autonaming (setting automatic
experiment name generation, the name contains
date and time of the experiment)
• Setting the ll mode for LV Unit use (for details see
chapter 2.12.1.1)
If module is selected additional commands (open/close
drawer) are available
Cleaning mode (opens the drawer to clean the retainer)
Shut down
321

4D-Nucleofector® Hardware Manual | 11
Figure 2.5: System settings screen (A) dropdown menu after tapping on the gear icon.
(B) Settings menu after tapping on “Settings” (Figure 2.5, A).
2.7.3 Turning off the 4D-Nucleofector® System
For a safe shut down of the system and protection against damage of
interior fragile parts, press the power button at the front of the Core
Unit and tap on “shutdown” on the screen. Alternatively, tap on the “set-
tings” icon (Figure 2.5, A) and then select “Shut Down”. Both ways the
4D-Nucleofector® System will shut down and the transportation lock
will be applied to delicate parts of the system.
Then switch off the device via the main power switch located at the
rear of the Core Unit. A hard shutdown of the system by pressing the
rear power switch of the Core Unit without a former software shutdown
should be avoided as this may lead to loss of data.
2.8 General instructions for running a
Nucleofection® Experiment
This chapter explains some general features and steps required to
perform a Nucleofection® Experiment independent of the different
4D-Nucleofector® Units. The detailed handling of each unit is described
in subsequent chapters 2.9 – 2.12.
A
B
2.8.1 Overview
Select vessel type
(chapter 2.8.2)
Dene new experiment
(chapters 2.9, 2.10, 2.11 or 2.12)
Saved user experiment
Modify
Load predened experiment
(chapter 2.8.4)
Lonza template
Use as is
Prepare sample according to cell-type specic Optimized Protocol
Load samples (chapters 2.9, 2.10, 2.11 or 2.12)
Execute Nucleofection® Experiment (chapter 2.9, 2.10, 2.11 or 2.12)
Figure 2.6: Schematic workflow overview of a typical Nucleofection® Experiment.
2.8.2 Unit and vessel selection
The main screen (Figure 2.7) shows all available vessels for attached
functional units (X, Y, 96-well, and LV Unit)
Figure 2.7: Example of main screen elements, depending on attached modules, up to
three functional modules can be attached to one Core Unit.
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12 | 4D-Nucleofector® Hardware Manual
By selecting the vessel type a new screen opens asking for the
experiment name. If “Experiment autonaming” is selected in the
settings menu (see chapter 2.7.2) a name is already entered and the
system opens the experimental setup screen directly after vessel
selection.
Figure 2.8: Start experiment by typing in the experiment name or click the “load” button
to load a predefined experiment (see chapter 8.4.2).
2.8.3 Experiment parameters – overview
An experiment comprises a selected set of vessels or wells that should
be processed with dened Nucleofection® Parameters.
To select a single well or a set of vessels simply touch the wells
displayed or drag along a set of vessels with your ngertip.
Figure 2.9: (A) Selecting single vessels by touching the screen at the respective well
position. (B/C) Multiple selection of vessels by dragging across the screen.
After selection there are several options for parameter denition.
Table 2.3 describes the parameters that can be dened.
Table 2.3: Entering Nucleofection® Conditions
Parameter Description
Sample type
(X ,Y and 96-well Unit)
1. Sample: This selection denes a normal sample
containing cells and substrate. The selected Nucleofec-
tion® Program will be applied to this position.
2. No DNA: Negative control. Nucleofection® Program
applied to vessel with cells but without substrate
3. No Prog.: 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 eect, i.e. no pulse will be applied.
Cell Type Code Using the “Cell Type Code” option, a “Pulse Code”
(e.g. FI-115) can be extended by the user with customized
cell-type codes (e.g., after a cell line optimization; 2.12.3).
Lonza dened programs are highlighted in blue while
custom program codes will be highlighted in black.
Solution In case the recommended Nucleofector® Program is not
selected automatically via the “Cell Type Code” option, the
“Solution” can be entered manually.
Pulse Code In case the recommended Nucleofector® Program is not
selected automatically via the “Cell Type Code” option, a
“PulseCode” (e.g. FI-115) can be entered manually.
Note: For manual “Pulse Code” selection, the “Solution”
has to be selected rst (see below).
Volume (LV Unit with
LV Nucleocuvette® Cartridge)
When using the scalable cuvette version, the
LV Nucleocuvette® Cartridge, the volume that should be
processed has to be dened. Depending on the number
of reservoirs used, one or two volume parameters must
be specied. The maximum total volume that can be
entered is 20 mL.
Fill mode (LV Unit with LV
Nucleocuvette® Cartridge)
When using the scalable cuvette version, the
LV Nucleocuvette® Cartridge, the ll mode (selected via
”Settings”) is indicated. It can be changed for an
individual experiment in the experiment screen.
A
B
C

4D-Nucleofector® Hardware Manual | 13
Figure 2.10: Setting up Nucleofection® Conditions.
2.8.4 Using a predefined experiment
A predened experiment can either be a ready-to-use template
pre-installed by Lonza (for X, Y, and 96-well Unit only) or a previous
experiment that was saved by the user.
The use of predened experiments is described showing the X Unit and
the 96-well Unit as examples, but the procedure is the same for Y or
LV Unit.
Figure 2.11: Selection of a pre-defined experiment
A
B
1. After vessel type selection (see 2.8.2) a screen appears prompting
to enter experiment name (Figure 2.9, A).
2. Selecting “Load” will open a list of experiments (Figure 2.9, B). The
list comprises experiments pre-dened by Lonza (template les;
blue) and all experiments saved by the user (black) for the selected
vessel type. Choosing an experiment and tapping the “Load” button
will open the experiments list specic for the vessel type selected.
Tapping on “Details” will provide information about name, vessel
type and number of dened vessels.
3. If you selected “Experiment autonaming” in the settings menu
(Chapter 2.7.2 and Table 2.2) you can load predened experiments
by tapping on the accordion menu (Figure 2.4 (1)) and select
“Experiments”. Tapping on “View” will show you the experimental
set up screen.
4. In case you want to modify the settings of a predened experiment,
click on the well(s) you want to change and adapt the settings
(for unit-specic details see chapters 2.9, 2.10, 2.11 or 2.12) once
loaded. You can save the changed experiments under a new name
by pressing “Save”. Alternatively, you are prompted to change the
name once you proceed to the summary screen (see next step)
and then save it. By tapping the “New” button you can name the
altered experiment and proceed.
5. To accept all settings, press “Next”.
6. The summary screen appears (Figure 2.12) listing the settings for
each well-dened and the drawer will open.
7. Load the samples (for unit- specic details see chapters 2.9.1,
2.10, 2.11 or 2.12) and press ”Start”
Figure 2.12: Summary screen after setup.
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14 | 4D-Nucleofector® Hardware Manual
2.9 4D-Nucleofector® X Unit
Figure 2.13: 4D-Nucleofector® Core and X Unit (A) and Nucleocuvette® Vessels and
Strips for the use with the X Unit (B).
The 4D-Nucleofector® X Unit can handle two types of vessels:
• 16-well 20 µL Nucleocuvette® Strips for cell numbers from 2 x 104to
1 x 106cells
• Single 100 µL Nucleocuvette ® Vessels for cell numbers from 2 x 105
to 2 x 107cells
By choosing the respective vessel type you will be directed to the
experiment setup screen.
A
B
Figure 2.14: Experiment naming (X Unit; example: 16-well Nucleocuvette® Strips); This
screen will only appear if you did not check “Experiment autonaming” in the settings
menu (Table 2.2)
2.9.1 Defining a new experiment
Following vessel type selection (see chapter 2.8.2) you can now
either select a predened experiment (see chapter 2.8.4) or dene an
experiment from the beginning.
If you want to create a new experiment, tap in the eld “Experiment
name” and enter an experiment name. If you selected “Experiment
autonaming” in the settings menu, the experiment name is
automatically generated with date and time stamp (see chapter 2.8.4).
You can change the name if you prefer to save it under a specic name
for later use. Tap on the checkmark button to get to Experiment Setup
screen (Figure 2.15).
A
B

4D-Nucleofector® Hardware Manual | 15
Figure 2.15: (A) Experiment setup screen for 16-well strip in 4D Nucleofector® X Unit,
Tapping on “Cell Type Code” will open a searchable screen for picking predefined Cell
Type Codes. (B) Scroll the screen, or use the arrows to navigate. (C) Use the search field
and start typing if you know the name of the Cell Type Code.
A screen appears with the title “Setup X Unit cuvette” or “Setup X Unit
16-well strip” (Figure 2.15, A) depending on your vessel selection.
1. Select well(s) to dene Nucleofection® Conditions
By tapping on a well position one sample/well is selected marked
by a blue circle around the well position. Multiple samples/wells can
be selected by dragging across the wells to select.
Tapping on additional wells will add more wells to the pre-selected
wells.
Remove a well from your selection by tapping on the selected
well again. Dragging across all selected wells will deselect
them, dragging across selected and deselected wells again will
toggle your selection. Alternatively, use “Deselect” to remove all
selections.
2. Choose your sample type
Pick “Sample” for samples which contain substrate and will be
pulsed
Pick “No DNA” for samples not containing any substrate (pulse
control)
Pick “No Pulse” for samples with substrate not being subjected to
an electrical pulse (no-pulse control)
3. Enter Cell Type Code (optional)
Press the eld “Cell Type Code” to choose predened Nucleofection®
Conditions from a cell type list (Figure 2.15, B). A list with all
available Cell Type Codes (dened by Lonza or custom) will appear.
Select the code that shall be used by scrolling through the menu or
start typing in the search eld (Figure 2.15, C).
To conrm your selection press “Apply”. If you pick a preset Cell
Type Code the next two steps do not apply
CNOTE: Instead of dening solution and program code via the Cell Type
Code, both parameters can also be selected manually, e.g. in case no
predened Cell Type Code is available. For adding new Cell Type Codes,
please refer to chapter 2.13.2.3.
4. Enter pulse code
If required, modify pulse code by pressing the pulse code eld. A
keyboard will appear, enabling you to change settings. Press the
“Apply” eld to conrm the selection.
5. The solution code can be modied via selecting the solution eld
and click on the desired solution. Then press “Apply” to conrm the
selection.
6. Optional: At this stage you can save your dened experiment or
only the pulse-solution code combination (as custom Cell Type
Code) for future use by pressing the “Save” button. Select whether
the Cell Type Code or the experiment will be saved. A keyboard will
appear allowing you to dene a name (max. length: 26 characters)
for either the Cell Type Code or the experiment depending on your
previous selection.
Conrm and save the experiment parameters by pressing “Next”
(saves the experiment and opens the drawer) or “Save” (saves the
experiment for later use).
Figure 2.16: Summary screen after experiment definition.
7. A summary of the dened settings will be displayed (Figure 2.16).
Please check for correctness before loading samples (see chapter
2.9.2) and starting the experiments by pressing “Start” (see
chapter 2.9.3)
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16 | 4D-Nucleofector® Hardware Manual
2.9.2 Loading samples
1. Load the 16-well Nucleocuvette® Strip (Figure 2.17, A, B) or one or
two 100 µL Nucleocuvette® Vessels into the sample tray (Figure
2.17, E, F)
2. Make sure that the strip is oriented properly:
For the 16-well Nucleocuvette® Strip, the yellow pin at the rear end
of the strip must be visible (Figure 2.17, C). If the strip is mounted
in the wrong orientation its rear end will stay above the strip holder
and the yellow pin is hardly visible (Figure 2.17, D).
For the 100 µL Nucleocuvette® Vessel, the cuvettes should be
mounted such that the label Lonza on the lid is readable and the
opening clip is at the front (Figure 2.17, G, H).
2.9.3 Running the experiment
Figure 2.18: Experiment progress (X Unit)
1. After loading the samples press “Start” to run the experiment
(Figure 2.16).
The progress of the experiment is indicated by changing the color
of the cuvette or well positions (Figure 2.18) (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 dened the drawer opens automatically after each set of two
and a message “Please insert cuvette for…” will be displayed. Load
next samples and press “OK” to continue or press “No” to interrupt the
experiment.
2. When the experiment is complete, a result le summarizing the
Nucleofection® Process will be displayed (Figure 2.19).
3. The result le will be saved automatically by the system (either as
Result + Date + Time or in case you saved the experiment under an
individual name as “Experiment name + Date + Time) and can be
reopened as described in chapter 2.13.2.2.
4. You can repeat the same experiment by pressing “Restart”.
5. Press “Restart” to start the experiment or “Finish” to return to home
screen.
Figure 2.19 Result Screen (A). Tap on wells to receive details about the experiment. For
explanation of symbols refer to chapter 4.1.
EF
G
A B
H
D Wrong orientationC Right orientation
Figure 2.17: Loading vessels into 4D Nucleofector® X Unit

4D-Nucleofector® Hardware Manual | 17
2.10 4D-Nucleofector® Y Unit
The Y Unit allows for transfection of cells in adherence. Cells plated in a
24-well dish are pulsed from the top with a specically designed dipping
electrode.
Figure 2.20: 4D-Nucleofector® Core and Y Unit and Dipping Electrode to use with the
Y Unit.
2.10.1 Defining a new experiment
Following vessel type selection (see chapter 2.8.2) you can now
either select a predened experiment (see chapter 2.8.4) or dene an
experiment from the beginning.
If you want to create a new Experiment, tap in the eld “Experiment
name” and enter a name. If you selected “Experiment autonaming” in
the settings menu, the experiment name is automatically generated
with date and time stamp (see chapter 2.8.4). (You can change the
name if you prefer by saving the experiment under a dierent name in
the next screen tapping on the “Save” button.) Press submit to get to
Experiment Setup screen (Figure 2.21).
A
B
Figure 2.21: Experiment setup screen for 24-well plate in 4D Nucleofector® Y Unit
A screen appears with the title “Setup Y Unit” (Figure 2.21).
1. Select well(s) to dene Nucleofection® Conditions
By tapping on a well position one sample/well is selected marked
by a blue circle around the well position. Multiple samples/wells can
be selected by dragging across the wells to select.
Tapping on additional wells will add more wells to the pre-selected
wells.
Remove a well from your selection by tapping on the selected
well again. Dragging across all selected wells will deselect
them, dragging across selected and deselected wells again will
toggle your selection. Alternatively, use “Deselect” to remove all
selections.
2. Choose your sample type
Pick “Sample” for samples which contain substrate and will be
pulsed
Pick “No DNA” for samples not containing any substrate (pulse
control)
Pick “No Pulse” for samples with substrate not being subjected to
an electrical pulse (no-pulse control)
3. Enter Cell Type Code (optional)
Press the eld “Cell Type Code” to choose predened Nucleofection®
Conditions from a cell type list (Figure 2.15, B). A list with all
available Cell Type Codes (dened by Lonza or custom) will appear.
Select the code that shall be used by scrolling through the menu or
start typing in the search eld (Figure 2.15, C).
To conrm your selection press “Apply”. If you pick a preset Cell
Type Code the next two steps do not apply
NOTE: Instead of dening solution and program code via the Cell Type
Code, both parameters can also be selected manually, e.g. in case no
predened Cell Type Code is available. For adding new Cell Type Codes,
please refer to chapter 2.13.2.3.
2

18 | 4D-Nucleofector® Hardware Manual
4. Enter pulse code
If required, modify pulse code by pressing the pulse code eld. A
keyboard will appear, enabling you to change settings. Press the
“Apply” eld to conrm the selection.
5. The solution code can be modied via selecting the solution eld
and click on the desired solution. Then press “Apply” to conrm the
selection.
Optional: At this stage you can save your dened experiment or
only the pulse-solution code combination (as custom Cell Type
Code) for future use by pressing the “Save” button. Select whether
the Cell Type Code or the experiment will be saved. A keyboard will
appear allowing you to dene a name (max. length: 26 characters)
for either the Cell Type Code or the experiment depending on your
previous selection.
6. Conrm and save the experiment parameters by pressing “Next”
(saves the experiment and opens the drawer) or “Save” (saves the
experiment for later use). The drawer will open allowing you to load
the sample and the summary screen will show the conditions for all
dened wells. Check for correctness.
2.10.2 Loading samples
Figure 2.22: Loading of samples (Y Unit). Watch the orientation of the plate. (Well A1 is
placed in the upper left position).
1. 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.
2. 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”.
3. It is not recommended to re-use dipping electrodes as this leads to
suboptimal transfection eciencies.
2.10.3 Running the experiment
1. After loading the samples press “Start” to run the experiment
2. The progress of the experiment is indicated by changing the color
of the cuvette or well positions (Figure 2.23A) (for color codes see
chapter 4).
3. When the experiment is complete, a result le summarizing the
Nucleofection® Process will be displayed (Figure 2.23, B).
4. The result le will be saved automatically by the system and can be
reopened as described in chapter 2.12.2.
5. Press “Restart” to start the experiment or “Finish” to return to home
screen.
Figure 2. 23: (A) Nucleofection® Progress (Y Unit). (B) Result Screen. Select wells to
receive result details.
A
B

4D-Nucleofector® Hardware Manual | 19
2.11 4D-Nucleofector® 96-well Unit
The 4D-Nucleofector® 96-well Unit is designed to simultaneously run up
to 96 Nucleofection® Reactions with cell numbers ranging from 2 x 104
to 1 x 106cells per reaction.
Figure 2.24: 4D-Nucleofector® Core Unit and 96-well Unit (A). 96-well Nucleocuvette®
Plate (B).
2.11.1 Defining a new experiment
Following vessel type selection (see chapter 2.8.2) you can now
either select a predened experiment (see chapter 2.8.4) or dene an
experiment from the beginning.
If you want to create a new experiment, tap in the eld “Experiment
name” and enter a name for the experiment. Tick the check mark icon to
reach the Experiment Setup screen (Figure 2.8).
A
B
Figure 2.25: Experiment definition (96-well Unit, plate view (A) and strip view(B))
1. Select well(s) to dene Nucleofection® Conditions
By tapping on a well position you can select one sample/well or you
can select multiple samples/wells at a time by dragging across the
wells to select.
Tapping on additional wells for addition of more wells to the pre-
selected wells.
Remove wells from your selection by tapping on the selected
well again. Dragging across all selected wells will deselect them,
dragging across selected and deselected wells again will toggle
your selection. Once a well is fully dened you will need to deselect
the well by tapping on the “Deselect” button in plate and strip view
when returning back from Setup window.
Switching from whole plate view (Figure 2.25 A) to strip view
(Figure 2.25B) is possible by tapping on the strip view/ plate
view button. In plate view mode select wells and tap on edit to
enter experiment conditions (Figure 2.26 A). In strip view further
information on the well position and applied pulses are visible for
each well. The selected positions are highlighted in blue (Figure
2.26B).
Tapping on the “Clear” button will delete all settings from the
selected wells after conrmation.
A
B
2

20 | 4D-Nucleofector® Hardware Manual
2. Choose your sample type
You can pick the regular sample for samples which contain
substrate and will be pulsed.
You can pick “No DNA” for samples not containing any substrate
(pulse control).
You can pick “No Pulse” for samples with substrate not being
subjected to an electrical pulse (no-pulse control)
3. Enter Cell Type Code (optional)
Press the eld “Cell Type Code” to choose predened Nucleofection®
Conditions from a cell type list (Figure 2.15). Enter the cell name in
the eld Cell Type Code.
To conrm your selection press “Apply”. If you pick a Cell Type Code
the next two steps do not apply
NOTE: Instead of dening solution and program code via the Cell Type
Code, both parameters can also be selected manually, e.g. in case no
predened Cell Type Code is available. For adding new Cell Type Codes,
please refer to chapter 2.13.2.3.
4. Enter pulse code
If required, modify pulse code by pressing the pulse code eld. A
keyboard will appear, enabling you to change settings. Press the
check mark eld to conrm the selection.
5. Enter Solution
The solution code can be modied via selecting the solution eld
and click on the desired solution. Then press “Apply” to conrm the
selection.
6. Save your experiment (optional)
At this stage you can save your dened experiment for future use
by pressing the “Save” button. You can choose to either save the
pulse code or the whole setup. A keyboard will appear allowing you
to dene a name (max. length: 26 characters). This step can be
ignored by pressing “Next”. The experiment is then automatically
saved.
7. Conrm and save the experiment parameters
By pressing “Next” (saves the experiment and opens the drawer)
the current settings will automatically be saved and a summary of
the dened settings will be displayed (Figure 2.27). Please check
for correctness before loading samples (see chapter 2.11.2) and
starting the experiments by pressing “Start”.
Figure 2.26 A: Experiment settings after well selection and tapping on “Edit” button in
plate view (A) and Sample definition in strip view (B).
For further explanation about parameters, please refer to chapter 2.8.3.
Figure 2.27: Summary screen after experiment setup.
A
B