Lonza AAF-1002X, AAF-1002Y, AAF-1003X Hardware Manual

Add to My manuals
40 Pages

advertisement

Lonza AAF-1002X, AAF-1002Y, AAF-1003X Hardware Manual | Manualzz
4D-Nucleofector
Hardware
Manual
Your guide to setup and usage of the
4D-Nucleofector® System
For research use only
®
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.
2 | 4D-Nucleofector® Hardware Manual
Content
1
Nucleofector® Technology
How it works
Transfection of any cell
Transfection of any substrate
Moving transfection into the fourth dimension
Experience the advanced Nucleofector® Platform:
4
4
4
4
5
5
2
Operating instructions
6
2.1
2.2
2.3
Restrictions
Maintenance
Safety instructions – please read carefully
2.3.1
Consignes de sécurité - à lire attentivement
2.4
2.5
2.6
2.7
Waste disposal
4D-Nucleofector® System components
Set-up instructions
General use instructions
8
9
10
10
2.7.1
2.7.2
2.7.3
Turning on the 4D-Nucleofector® System
Operating software
Turning off the 4D-Nucleofector® System
10
10
11
2.8
General instructions for running a Nucleofection® Experiment
11
2.8.1
2.8.2
2.8.3
2.8.4
Overview
Unit and vessel selection
Experiment parameters – overview
Using a predefined experiment
11
11
12
13
2.9
4D-Nucleofector® X Unit
14
2.9.1
2.9.2
2.9.3
Defining a new experiment
Loading samples
Running the experiment
14
16
16
2.10
4D-Nucleofector® Y Unit
17
2.10.1
2.10.2
2.10.3
Defining a new experiment
Loading samples
Running the experiment
17
18
18
2.11
4D-Nucleofector® 96-well Unit
19
2.11.1
2.11.2
2.11.3
Defining a new experiment
Loading samples
Running the experiment
19
21
21
6
6
6
7
2.12
4D-Nucleofector® LV Unit
22
2.12.1
2.12.1.1
2.12.1.2
2.12.1.3
2.12.2
2.12.2.1
2.12.2.2
2.12.2.3
2.12.2.4
Using the 1 mL Nucleocuvette® Cartridge (Fixed volume)
Defining a new experiment
Loading Samples
Running the experiment
Using the LV Nucleocuvette® Cartridge (Scalable volume)
Defining a new experiment
Mounting an LV Nucleocuvette® Cartridge
Loading Samples
Running the experiment
23
23
24
24
25
26
26
28
30
2.13
Additional settings and options
31
2.13.1
2.13.1.1
2.13.1.2
2.13.2
2.13.2.1
2.13.2.2
2.13.2.3
2.13.2
2.13.4
Settings menu on the right
System settings
Cleaning the Nucleofection® Vessel Tray (X, Y, or 96-well Unit)
Left menu
E xperiments
Results
Custom Cell Type Codes
Lonza programs
Firmware update
31
31
31
32
32
32
32
33
33
3
Troubleshooting
3.1
3.2
Suboptimal transfection results
Rescue of samples (X and Y Unit)
4
Error codes of the
4D-Nucleofector® System
4.1
4.2
4.3
Color coding of wells
Error codes
Arc discharge correction
5
Purchaser notification
Purchaser notification
Representations and warranties
6
Technical specifications
34
34
35
36
36
36
37
37
37
37
39
4D-Nucleofector® Hardware Manual | 3
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 reflect 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 specifically optimized
electrical parameters and Nucleofector® Kits, which contain specific
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 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 difficult-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 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® Technology 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.
www.lonza.com/celldatabase
4 | 4D-Nucleofector® Hardware Manual
1
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 offer advanced performance, flexibility and
convenience. The system is controlled by intuitive software on an upto-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 different cell numbers using the same conditions
• Nucleofection® Conditions for cells in adherence
• Closed, scalable transfection of larger cell numbers in the range of
1x107 to 1x109 cells per reaction
Scientific Support Teams
Europe
Phone: + 49 221 99199 400
E-mail: [email protected]
North America
Phone: 800 521 0390 (toll free)
E-mail: [email protected]
lonza.com/transfection
4D-Nucleofector® Hardware Manual | 5
2 Operating instructions
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.
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 figure 2.1). Blown fuses can easily be replaced. Disconnect the
4D-Nucleofector® System 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.
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 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-profit-entities, as set forth in the license.
For license information contact Lonza Cologne GmbH:
Phone: + 32 87 321 611
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. 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.
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
identified 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 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 cells.
These electrical impulses can be deadly!
6 | 4D-Nucleofector® Hardware Manual
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
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 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.
2.3.1 Consignes de sécurité - à lire attentivement
Ce symbole signifie qu‘il existe un risque de choc électrique. Un choc
électrique peut provoquer des blessures graves, voire mortelles.
L’appareil 4D-Nucleofector® a été certifié 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.
4D-Nucleofector® Hardware Manual | 7
2
• 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
8 | 4D-Nucleofector® Hardware Manual
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, assurezvous 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.
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. 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).
2
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:
X Unit:
Y Unit:
LV Unit:
96-well:
AAF-1003B
AAF-1003X
AAF-1003Y
AAF-1002LW
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:
X Unit:
Y Unit:
LV Unit:
96-well:
9**B2***
9**X2***
9**Y2***
9**LV2**
9**S2***
Unit
arrangement
Horizontal or vertical
(via demountable
side panels)
Vertical only
(fixed side panels)
Silver
Core Unit –
display frame
Blue
Blue
X/Y Unit – sample rescue using wheel accessible after removal of side panel via screw
behind termination plug (also see 3.2)
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)
A typical 4D-Nucleofector® System includes (Figure 2.2 and
Figure 2.3 A and B):
1
2
3
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 109 cells
4
6
5
7
11
8
10
12
9
Figure 2.3: (A) 4D-Nucleofector® Core Unit, (B) 4D-Nucleofector® System comprising of
Core, X, and 96-well Unit in rear view.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Power cord socket with main power switch and fuses
Power cord
USB port(s)
Lonza interface outlet port to connect functional units
Lonza interface inlet port to connect functional units
Lonza interface inlet port of last functional unit
Lonza interface cable connected to outlet and inlet port
Lonza interface outlet port of functional unit
Lonza outlet port of last functional unit with termination plug
4D-Nucleofector® Hardware Manual | 9
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 first 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 first
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
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.
1
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, figure 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.
10 | 4D-Nucleofector® Hardware Manual
3
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 specific 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 different
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 fill 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
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 specific Optimized Protocols
(https://knowledge.lonza.com).
2
2.8.1
A
Overview
Select vessel type
(chapter 2.8.2)
Load predefined experiment
(chapter 2.8.4)
B
Lonza template
Use as is
Define new experiment
(chapters 2.9, 2.10, 2.11 or 2.12)
Saved user experiment
Modify
Prepare sample according to cell-type specific Optimized Protocol
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 “settings” 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
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)
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.
Figure 2.7: Example of main screen elements, depending on attached modules, up to
three functional modules can be attached to one Core Unit.
4D-Nucleofector® Hardware Manual | 11
2
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.
C
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 definition.
Table 2.3 describes the parameters that can be defined.
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 defined Nucleofection® Parameters.
Table 2.3: Entering Nucleofection® Conditions
Parameter
Description
Sample type
(X ,Y and 96-well Unit)
1. Sample: This selection defines a normal sample
containing cells and substrate. The selected 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 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 effect, 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 defined 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 first (see below).
To select a single well or a set of vessels simply touch the wells
displayed or drag along a set of vessels with your fingertip.
A
B
When using the scalable cuvette version, the
Volume (LV Unit with
LV Nucleocuvette® Cartridge) LV Nucleocuvette® Cartridge, the volume that should be
processed has to be defined. Depending on the number
of reservoirs used, one or two volume parameters must
be specified. The maximum total volume that can be
entered is 20 mL.
Fill mode (LV Unit with LV
Nucleocuvette® Cartridge)
12 | 4D-Nucleofector® Hardware Manual
When using the scalable cuvette version, the
LV Nucleocuvette® Cartridge, the fill mode (selected via
”Settings”) is indicated. It can be changed for an
individual experiment in the experiment screen.
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-defined by Lonza (template files;
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 specific for the vessel type selected.
Tapping on “Details” will provide information about name, vessel
type and number of defined vessels.
Figure 2.10: Setting up Nucleofection® Conditions.
2.8.4
Using a predefined experiment
A predefined 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 predefined 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.
A
3. If you selected “Experiment autonaming” in the settings menu
(Chapter 2.7.2 and Table 2.2) you can load predefined 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 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, 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-defined and the drawer will open.
7.
Load the samples (for unit- specific details see chapters 2.9.1,
2.10, 2.11 or 2.12) and press ”Start”
B
Figure 2.12: Summary screen after setup.
Figure 2.11: Selection of a pre-defined experiment
4D-Nucleofector® Hardware Manual | 13
2
2.9
4D-Nucleofector® X Unit
A
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)
B
2.9.1
Defining a new experiment
Following vessel type selection (see chapter 2.8.2) you can now
either select a predefined experiment (see chapter 2.8.4) or define an
experiment from the beginning.
If you want to create a new experiment, tap in the field “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 specific name
for later use. Tap on the checkmark button to get to Experiment Setup
screen (Figure 2.15).
A
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 104 to
1 x 106 cells
• Single 100 µL Nucleocuvette ® Vessels for cell numbers from 2 x 105
to 2 x 107 cells
By choosing the respective vessel type you will be directed to the
experiment setup screen.
14 | 4D-Nucleofector® Hardware Manual
B
NOTE: Instead of defining solution and program code via the Cell Type
Code, both parameters can also be selected manually, e.g. in case no
predefined Cell Type Code is available. For adding new Cell Type Codes,
please refer to chapter 2.13.2.3.
C
4. Enter pulse code
If required, modify pulse code by pressing the pulse code field. A
keyboard will appear, enabling you to change settings. Press the
“Apply” field to confirm the selection.
5. The solution code can be modified via selecting the solution field
and click on the desired solution. Then press “Apply” to confirm the
selection.
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.
6. Optional: At this stage you can save your defined 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 define a name (max. length: 26 characters)
for either the Cell Type Code or the experiment depending on your
previous selection.
1. Select well(s) to define Nucleofection® Conditions
Confirm and save the experiment parameters by pressing “Next”
(saves the experiment and opens the drawer) or “Save” (saves the
experiment for later use).
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)
Figure 2.16: Summary screen after experiment definition.
7.
A summary of the defined 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)
3. Enter Cell Type Code (optional)
Press the field “Cell Type Code” to choose predefined Nucleofection®
Conditions from a cell type list (Figure 2.15, B). A list with all
available Cell Type Codes (defined by Lonza or custom) will appear.
Select the code that shall be used by scrolling through the menu or
start typing in the search field (Figure 2.15, C).
To confirm your selection press “Apply”. If you pick a preset Cell
Type Code the next two steps do not apply
4D-Nucleofector® Hardware Manual | 15
2
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)
2.9.3
Running the experiment
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).
A
B
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).
C Right orientation
D Wrong orientation
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 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 file summarizing the
Nucleofection® Process will be displayed (Figure 2.19).
E
F
3. The result file 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.
G
H
Figure 2.17: Loading vessels into 4D Nucleofector® X Unit
Figure 2.19 Result Screen (A). Tap on wells to receive details about the experiment. For
explanation of symbols refer to chapter 4.1.
16 | 4D-Nucleofector® Hardware Manual
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 specifically designed dipping
electrode.
A
2
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 define 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.
B
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
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 predefined experiment (see chapter 2.8.4) or define an
experiment from the beginning.
If you want to create a new Experiment, tap in the field “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 different name in
the next screen tapping on the “Save” button.) Press submit to get to
Experiment Setup screen (Figure 2.21).
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 field “Cell Type Code” to choose predefined Nucleofection®
Conditions from a cell type list (Figure 2.15, B). A list with all
available Cell Type Codes (defined by Lonza or custom) will appear.
Select the code that shall be used by scrolling through the menu or
start typing in the search field (Figure 2.15, C).
To confirm your selection press “Apply”. If you pick a preset Cell
Type Code the next two steps do not apply
NOTE: Instead of defining solution and program code via the Cell Type
Code, both parameters can also be selected manually, e.g. in case no
predefined Cell Type Code is available. For adding new Cell Type Codes,
please refer to chapter 2.13.2.3.
4D-Nucleofector® Hardware Manual | 17
4. Enter pulse code
If required, modify pulse code by pressing the pulse code field. A
keyboard will appear, enabling you to change settings. Press the
“Apply” field to confirm the selection.
5. The solution code can be modified via selecting the solution field
and click on the desired solution. Then press “Apply” to confirm the
selection.
Optional: At this stage you can save your defined 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 define a name (max. length: 26 characters)
for either the Cell Type Code or the experiment depending on your
previous selection.
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 file summarizing the
Nucleofection® Process will be displayed (Figure 2.23, B).
4. The result file 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.
A
6. Confirm 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
defined wells. Check for correctness.
2.10.2
Loading samples
B
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 efficiencies.
18 | 4D-Nucleofector® Hardware Manual
Figure 2. 23: (A) Nucleofection® Progress (Y Unit). (B) Result Screen. Select wells to
receive result details.
2.11 4D-Nucleofector® 96-well Unit
A
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 106 cells per reaction.
A
2
B
B
Figure 2.25: Experiment definition (96-well Unit, plate view (A) and strip view(B))
1. Select well(s) to define 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.
Figure 2.24: 4D-Nucleofector® Core Unit and 96-well Unit (A). 96-well Nucleocuvette®
Plate (B).
Tapping on additional wells for addition of more wells to the preselected wells.
2.11.1 Defining a new experiment
Following vessel type selection (see chapter 2.8.2) you can now
either select a predefined experiment (see chapter 2.8.4) or define an
experiment from the beginning.
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 defined you will need to deselect
the well by tapping on the “Deselect” button in plate and strip view
when returning back from Setup window.
If you want to create a new experiment, tap in the field “Experiment
name” and enter a name for the experiment. Tick the check mark icon to
reach the Experiment Setup screen (Figure 2.8).
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 confirmation.
4D-Nucleofector® Hardware Manual | 19
2. Choose your sample type
You can pick the regular sample for samples which contain
substrate and will be pulsed.
A
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 field “Cell Type Code” to choose predefined Nucleofection®
Conditions from a cell type list (Figure 2.15). Enter the cell name in
the field Cell Type Code.
To confirm your selection press “Apply”. If you pick a Cell Type Code
the next two steps do not apply
B
NOTE: Instead of defining solution and program code via the Cell Type
Code, both parameters can also be selected manually, e.g. in case no
predefined 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 field. A
keyboard will appear, enabling you to change settings. Press the
check mark field to confirm the selection.
5. Enter Solution
The solution code can be modified via selecting the solution field
and click on the desired solution. Then press “Apply” to confirm the
selection.
6. Save your experiment (optional)
At this stage you can save your defined 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 define a name (max. length: 26 characters). This step can be
ignored by pressing “Next”. The experiment is then automatically
saved.
7.
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.
Confirm 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 defined 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 2.27: Summary screen after experiment setup.
20 | 4D-Nucleofector® Hardware Manual
2.11.2 Loading samples
Load the 96-well Nucleocuvette® Plate (Figure 2.24) into the sample
tray (Figure 2.28)
Selecting a well will display further information of the result. Tapping the
“Details” button will open a description for this well, including details
about the setup and result.
A
2
B
Figure 2.28: 96-well Unit sample tray. Watch the orientation of the plate. (Well A1 is
placed in the upper left position).
Make sure that the plate is oriented properly: the plate needs to be
oriented in a way that well A1 is placed in the upper left corner.
2.11.3 Running the experiment
After loading the samples press “Start” to run the experiment
(Figure 2.27).
The progress of the experiment is indicated by changing the symbols of
the well positions.
When the experiment is complete, a result file summarizing the
Nucleofection® Process will be displayed (Figure 2.28).
Figure 2.30: Detail screen for experiment results. (A) Selection of well is indicated by
blue circle. (B) Clicking on “Details” button reveals further information on the well.
You can repeat the same experiment by pressing “Restart” leading to the
summary screen. Or tap on “Finish” to leave this experiment.
Press “Start” to start the same experiment again or “Back” to return to
the “Set up Experiment” screen and define a new experiment
Figure 2.29: Example of a result file.
The result file will be saved automatically by the system and can be
reopened as described in chapter 2.13.2.2.
4D-Nucleofector® Hardware Manual | 21
2.12 4D-Nucleofector® LV Unit
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.
A
4
1
Components of the LV Nucleocuvette® Cartridge
(Figure 2.31, B, C)
1. Cartridge
2. Venting tube with 0.2 µM sterile filter
3. Inlet port 1 (spinning Spiros with red dust protection cap)
4. Inlet port 2 (spinning Spiros with red dust protection cap)
5. Outlet port (spinning Spiros with red dust protection cap)
6. Reservoirs (swabbable injection port with male Luer cap)
NOTE: Please do not alter or disassemble the pre-assembled cartridge
or reservoirs.
D
2
3
4
6
3
Components of 1 mL Nucleocuvette® Cartridge
(Figure 2.31, A)
1. Transparent handle/handle bar
2. Contacting side/part/area
3. Injection port (rear side)
4. Air outlet port with 0.2 µM sterile filter
2
5
B
3
4
5
1
2
C
6
22 | 4D-Nucleofector® Hardware Manual
1
Components of the LV Nucleocuvette® Cartridge, weldable tubing
(Figure 2.31, D)
1. Cartridge
2. Venting tube with 0.2 μM sterile filter
3. Inlet port 1 (spinning Spiros with red dust protection cap)
4. Inlet port 2 (spinning Spiros with red dust protection cap)
5. Outlet port (spinning Spiros with red dust protection cap)
6. PVC tube attachments with clamps (for welding)
Figure 2.31: Nucleocuvette® Cartridge types for LV Unit
2.12.1 Using the 1 mL Nucleocuvette® Cartridge (Fixed volume)
2.12.1.1 Defining a new experiment
Following vessel type selection (see chapter 2.8.2) you can now
either select a predefined experiment (see chapter 2.8.4) or define an
experiment from the beginning.
If you want to create a new experiment, tap in the field “Experiment
name” and enter a name for the experiment. Tick the check mark icon to
reach the Experiment Setup screen (Figure 2.8).
2
A
Figure 2.33: List of predefined Cell Type Codes. Select the Code by either typing in a
name in the Search Field, scrolling the screen or using the arrows on the right of the
screen.
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
2. To confirm your selection press “Apply”.
3. 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.
Instead of defining solution and program code via the Cell Type Code,
both parameters can also be selected manually, e.g. in case no
predefined Cell Type Code is available. For adding new Cell Type Codes,
please refer to chapter 2.13.2.3.
4. Optional: At this stage you can save your defined experiment
for future use by pressing the “Save” button (Figure 2.34, A). A
keyboard will appear allowing you to define a name (max. length:
26 characters, figure 2.34, B).
Figure 2.32: (A) Experiment setup screen for 4D-Nucleofector® LV Unit using 1 mL
Nucleocuvette® Cartridge. (B) Progress Screen during experiment.
A screen appears with the title “Setup LV 1 mL” (Figure 2.32, A).
1. Press the field “Cell Type Code” to choose predefined Nucleofection®
Conditions from a cell type list (Figure 2.33). Sort list functions
(A-Z) to find conditions more quickly by using the scroll bar at the
side.
4D-Nucleofector® Hardware Manual | 23
A
2.12.1.2 Loading Samples
1. Fill the 1 mL Nucleocuvette® Cartridge with the sample. For filling
hold cartridge in a slight angle as shown in figure 2.35 (A). The
sterile filter on the rear side should be at the top, the injection port
should be at the bottom (upon pipetting cartridge will be filled from
bottom to top). Insert the pipette tip approximately in a 90° angle.
A
B
B
Figure 2.35: A volume of 1 mL is required to completely fill the cuvette. Using a higher
or lower volume may lead to errors.
2. Insert the filled cartridge into the slot until it snaps in (Figure
2.35, B). Make sure that upper and lower snap mechanism audibly
engages to allow for proper contacting.
C
Figure 2.34: Saving an experiment. (A) after tapping the “Save” icon in the setup screen.
Choose between Cell Type Code or Experiment. (B) Type in the name for Either Experiment or Cell Type Code. (C) Click “OK” to proceed.
5.
Confirm and save the experiment parameters by pressing “OK” or
“SAVE”.
6. Press “OK” to continue (Figure 2.34, C)
7. A summary of the defined settings will be displayed. Please check
for correctness before continuing.
24 | 4D-Nucleofector® Hardware Manual
2.12.1.3 Running the experiment
1. When the cartridge is inserted, press “Next” to initiate the
Nucleofection® Process.
2. The progress of the experiment is indicated by a progress bar
(Figure 2.32, B).
3. When the experiment is complete, a result file summarizing the
Nucleofection® Process will be displayed.
4. The result file 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.
5. The overall result is indicated by a color code:
a. Green cross for “OK” (Figure 2.36, A)
b. Yellow cross in case of few errors (Figure 2.36, B)
c. Red dash in case of multiple errors (not shown).
A
If errors occurred you can check for more details by pressing “Details”.
The result details (Figure 2.36) will display the type of error and its’
percent occurrence in the processed volume. For additional information
on the error press “Details”.
6. Once the process is completed take out the cartridge by pressing
the black levers.
7. Aspirate the transfected sample via the port (see figure 2.35, A)
and transfer cells into culture.
Optionally you may wash the cartridge with medium to aspirate
residual liquid.
8. You can repeat the same experiment by pressing “Restart”.
B
C
2.12.2 Using the LV Nucleocuvette® Cartridge (Scalable volume)
For running an experiment with the LV Nucleocuvette® Cartridge the
recommended order of steps is:
Step 1: Start defining the experiment (see 2.12.2.1)
Step 2: Mount the LV Nucleocuvette® Cartridge including all tubes onto
the 4D-Nucleofector® LV Unit (see 2.12.2.1).
NOTE: In case you prefer establishing the connection of the LV
Nucleocuvette® Cartridge to the input and output reservoirs
prior to cartridge mounting at the device, please make sure the
tubings are clamped to avoid liquid movement.
Step 3: Connect LV Nucleocuvette® Cartridge and output LV Reservoir
(see 2.12.2.2) add medium to the output reservoir, insert it into
a 4D-Nucleofector® LV Rack and connect it to the outlet tube of
the LV Nucleocuvette® Cartridge (see steps 11–1, see page 28)
either by manual connection or by welding (when using the LV
Nucleocuvette® Cartridge with weldable tubing)
Step 4: Prepare your cell sample and substrate using the input LV
Reservoirs (see 2.12.2.3)
Step 5: Connect the input LV Reservoirs to LV Nucleocuvette® Cartridge
(see 2.12.2.3)
Step 6: Verify volume settings and execute experiment (see 2.12.2.3)
NOTE: In case you use bags and not Lonza LV Reservoirs for sample
input and/or collection, please contact Lonza Scientific Support for
maximum tube volumes.
D
Figure 2.36: Result screens for LV Unit. (A/B) Result Screen for no errors in experiment. (C/D) Result Screen for experiment with few errors. Tapping the “Details” icon will
provide further information on the pulse delivery.
4D-Nucleofector® Hardware Manual | 25
2
processing until the end of liquid according to volume entry
is reached or if multiple errors occur that point to missing
liquid.
2.12.2.1 Defining a new experiment
Following vessel type selection (see chapter 2.8.2) you can now
either select a predefined experiment (see chapter 2.8.4) or define an
experiment from the beginning.
If you want to create a new experiment, tap in the field “Experiment
name” and enter a name for the experiment. Tick the check mark icon to
reach the Experiment Setup screen (Figure 2.8).
1. Enter Cell Type Code (optional)
Press the field “Cell Type Code” to choose predefined Nucleofection®
Conditions from a cell type list (Figure 2.33). Enter the cell name in
the field “Cell Type Code”.
To confirm your selection press “Apply”. If you pick a Cell Type Code
the next two steps do not apply
NOTE: Instead of defining solution and program code via the Cell
Type Code, both parameters can also be selected manually (step
2 and 3), e.g. in case no predefined Cell Type Code is available. For
adding new Cell Type Code, please refer to chapter 2.13.2.3.
2. Enter pulse code
If required, modify pulse code by pressing the pulse code field. A
keyboard will appear, enabling you to change settings. Press the
check mark field to confirm the selection.
3. Enter Solution
The solution code can be modified via selecting the solution field
and click on the desired solution. Then press “Apply” to confirm the
selection.
4. Enter the volume you plan to process
Press on the field “Volume” (Cell suspension volume) to define the
planned volume of your cell suspension (up to 20 mL are possible).
a. If you work with one reservoir (or bag) containing a premixed
suspension of cells and substrate, enter the total volume of the
cell suspension you want to process.
b. In case you work with two reservoirs (or bags) to keep cells
and substrate separate, tick the box “Substrate separate”.
A second volume field will show up for “Substrate” to define
the volume of substrate independently. In addition a field will
appear named “Bubble detection”:
• If bubble detection is de-activated (default setting), the
liquid sensor is only used for priming of the substrate line
(detection of liquid start). Afterwards the system would
ignore any air fractions within the substrate feed and
continue processing until the end of liquid according to
volume entry is reached or if multiple errors occur that
point to missing liquid. However, the air fraction may lead
to an increased error frequency. In addition larger fractions
without substrate may lead to a decrease in transfection
efficiency, as the sample is pulsed without substrate.
• If bubble detection is activated, the system will interrupt the
pulsing process in case the substrate line contains a larger
bubble (>7 mm). A message will appear and the user can
decide whether the process shall be continued or aborted.
In case “continue” is selected, the system will continue the
process like in the “de-activated” setting, i.e. it would ignore
any further air fractions in the substrate feed and continue
26 | 4D-Nucleofector® Hardware Manual
NOTE: The maximum total volume that can be entered is 20 mL and the
ratio between Cell suspension and Substrate volume should be between
1:1 (e.g. 10 mL cell suspension and 10 mL substrate) and 9:1
(e.g. 18 mL cell suspension and 2 mL substrate)
5. Save your experiment (optional)
At this stage you can save your defined 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 define a name (max. length: 26 characters). This step can be
ignored by pressing “Next”. The experiment is then automatically
saved.
6. Continue with mounting the LV Nucleocuvette® Cartridge
(see 2.12.2.2)
2.12.2.2 Mounting an LV Nucleocuvette® Cartridge
1. Insert the LV Nucleocuvette® Cartridge into the slot. Make sure that
upper and lower snap mechanism audibly engages to allow for
proper contacting.
2. Attach the venting tube with the filter (upper left of the cartridge) to
the male Luer connector on the front plate (Figure 2.37, A).
A
Figure 2.37: (A) Mounting the LV Nucleocuvette® Cartridge.*
*The picture shown for 2.37: (A) is the first version of the consumable, later versions
may show slight modifications. However, the mounting procedure stays the same.
3. If you started defining your experiment setup already (see
2.12.2.1), the software can support you in inserting the tubes into
the pinch valves by automatic opening of the valves. On the right
side of the experiment screen you can select “Yes” if you want to
insert the tubes of the LV Nucleocuvette® Cartridge into the pinch
valves now (Figure 2.37, B). Alternatively, you can open the pinch
valves manually by pushing the black button on the valves.
4. Upon pressing “Yes” the pinch valves will automatically open for
30 seconds allowing to conveniently insert the tubes into the three
pinch valves (Figure 2.37, C).
NOTE: The open time of the pinch valves is limited to 30 seconds to
avoid overloading of the electronics.
B
D
2
Figure 2.37: (D) Insertion into three pinch valves.*
E
Figure 2.37: (E) Complete insertion into three pinch valve.*
Figure 2.37: (B) Setup Screen for 4D-Nucleofector® LV Cartridge.
C
5. Insert the tubes into the three pinch valves (Figure 2.37, D).
Ensure that they are inserted completely (Figure 2.37, E). After
30 seconds, the pinch valves will close automatically. In case you
did not finalize insertion within 30 seconds, you may re-open the
valves by pressing “YES” again or manually (see “Note” above).
6. Insert the T connector on the left hand side of the cartridge into the
T connector holder (Figure 2.37, F, left).
Figure 2.37: (C) The remaining time for inserting the tubes is shown. If the time is not
sufficient you can start the process again after the time ran up.
*The picture shown for 2.37: (D/E) is the first version of the consumable, later versions
may show slight modifications. However, the mounting procedure stays the same.
4D-Nucleofector® Hardware Manual | 27
F
H
I
Figure 2.37: (F) insertion into the T connector holder and liquid sensors.*
7.
Insert the two larger diameter tubes into the black holders
(figure2.37, F, right). Ensure the tubes are inserted completely into
the black holders. The lower holder comprises a light sensor for
liquid detection in the lower tubing line.
G
Figure 2.37: (G) Insertion in upper pump.*
8. Insert the upper tube into the upper pump and close the flap
(Figure 2.37, G). When closing the flap, gently hold the tube without
pulling it.
9. Repeat with the lower tube and the lower peristaltic pump (optional,
if working with 2 input reservoirs).
10. Fill the output reservoir with medium (for detailed
recommendations, please refer to cell type-specific protocol) and
mount it into a 4D-Nucleofector® LV Reservoir Rack (Figure 2.37, H).
11. Remove the red cap from the Spiros connector on the outlet tube of
the cartridge and connect it to the swabbable injection port of the
output reservoir (or any other reservoir with female Luer connector,
figure 2.37, I). Alternatively, when working with the weldable
LV cartridge version, you may establish the connection by welding.
12. Before continuing check setup status: At this point steps 1–6 and
8 of the arrangement shown in figure 2.31 should be done.
13. Continue with sample loading (see 2.12.2.3).
*The picture shown for 2.37: (F/G/H/I/J) is the first version of the consumable, later versions may show slight modifications. However, the mounting procedure stays the same.
28 | 4D-Nucleofector® Hardware Manual
Figure 2.37: (H) Reservoir filled with medium and mounted into a 4D Nucleofector® LV
Reservoir Rack. (I) Luer connector.*
2.12.2.3 Loading Samples
1. Prepare cell suspension under the sterile hood (for detailed
recommendations, please refer to cell type-specific protocol).
2. Fill a defined volume of cells and substrate into the input
reservoir(s) mounted on a 4D-Nucleofector® LV Reservoir Rack.
3. Place the rack with the cell suspension reservoir on a magnetic
stirring platform to avoid cell sedimentation when working with
larger volumes. Start stirring the cell suspension at ~300 rpm.
Ensure that magnet is truly stirring.
4. Remove red caps from the Spiros connectors on the inlet tubes
of the cartridge and the blue caps from the swabbable injection
port of the output reservoirs and connect both (Figure 2.37, G).
Alternatively, when working with the weldable LV cartridge version,
you may establish the connection by welding.
5. The system is now fully assembled. Check correct assembly as
shown in figure 2.38.
2
8a
2
7
3
5
4
6
1
8b
8c
1. Cartridge inserted in slot
2. Short venting tube with filter attached to the male Luer connector
on the front plate
3. Tubes inserted in pinch valves (press black round surface of the
valves while pulling tubes fully into the valves)
4. T connector inserted into T connector holder. Two larger diameter
tubes completely inserted into liquid sensors
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.
7. Holder for cell suspension reservoir placed on a magnetic
stirring platform (stirring speed to approx. 300 rpm)
8. All reservoir-tube connections established to
a. Input reservoir for cell suspension
b. Input reservoir for substrate (optional)
c. Output reservoir
Figure 2.38: Setup Checklist for LV Nucleocuvette® Cartridge.
*The picture shown for 2.38: is the first version of the consumable, later versions may
show slight modifications. However, the mounting procedure stays the same.
4D-Nucleofector® Hardware Manual | 29
2.12.2.4 Running the experiment
1. Verify again that the volumes you have entered before (see
2.12.2.1, step 5) correctly reflects the volumes you have prepared.
If not, adapt volumes again.
2. Upon pressing “Next” a summary of the defined settings will be
displayed. Please check for correctness before pressing “Start” to
initiate the Nucleofection® Process.
3. Press “Start”. The system will remind you to open the clamps
(Figure 2.39, A).
A
A
B
B
Figure 2.40: (A) Result screen. (B) Result details after tapping on the “Details” Button
(Figure 2.32 A)
7.
8.
Figure 2.39: (A) Prompt before starting the actual experiment to check tubing and open
clamps. (B) Progress of the experiment.
4. Before starting the liquid priming process, the system performs a
calibration step.
5. The progress of the experiment is indicated by a progress bar
(Figure 2.39, B).
6. Once the required cycles to process the sample are completed,
a result screen summarizing the Nucleofection® Process will be
displayed (Figure 2.39, A).
30 | 4D-Nucleofector® Hardware Manual
9.
10.
11.
12.
The result file 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.
The overall result is indicated by a color code:
• Green cross for “OK”
• Yellow cross in case of few errors (Figure 2.40, A)
• Red dash in case of multiple errors
If errors occurred you can check for more details by pressing
“Details”. The result details (Figure 2.40, B) will display the type
of error and its’ percent occurrence in the processed volume. For
additional information on the error press “Details”.
Once the process is completed disconnect the output reservoir and
transfer transfected cell suspension into cell culture vessels.
Disassemble the tubing, take out the LV Nucleocuvette® Cartridge
by pressing the black levers (Figure 2.37, I) and discard it.
You can repeat the same experiment by pressing “Restart”.
Press “Start” to start the experiment again, or “Back” return to the
“Experiment set up” screen.
2.13 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.
2.13.1 Settings menu on the right
Activate the “Settings” menu by tapping on the gear icon on the right on
the home screen (Figure 2.4).
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.
A screen appears displaying a list of setting options (Figure 2.41).
A
B
Figure 2.41: Settings menu without (A) or with (B) upfront choice of functional unit.
Tap on the “Settings” field to open the settings screen
2.13.1.1 System settings
• Current time: Set or change the time by tapping on this field.
• Time format: Switch between 24 h or AM/PM format
• Current Date: Tap on this field to adjust the date
• Date format: Tapping on this field allows for setting different date
formats
• Transportation mode: This field is checked by default and will apply
the transportation lock upon shutdown of the system.
• Experiment autonaming: Once checked experiment names are
generated automatically in the format “Exp_yyyy-MMM-dd_hhmm-ss”. The screen for naming the experiment (Figure 2.8) is
not displayed. Changing this name is possible after setup of the
experiment and save experiment (Figure 2.34)
• Brightness: Use the slider to adjust brightness of the screen.
• Volume: Use slider or tap on the speaker icon to adjust or mute
sound.
• Network: Shows available network connections
• Version: Displays software and firmware versions of all connected
4D-Nucleofector® Units.
• Optional Fill mode (only available if LV Unit is connected): The
“Fill mode” can be adapted when using the flow-through cartridge
version, the LV Nucleocuvette® Cartridge. The standard fill mode “1”
calculates with a slight overfill of the cartridge to compensate for
variations in tube diameters and avoid an underfill, as this can lead
to arcing when using strong pulses. Since this “overfill” strategy
may also lead to some potential dead volume, the filling strategy can
be adapted for an individual use case by selecting other fill modes
from “2” (moderate reduction of overfill) to “5” (strong reduction
of overfill). This allows the user to select the standard fill mode (1)
which safely avoids underfill and arcing or try different underfill
levels to maximize the transfected volume (knowing that this may
cause arcing).
2.13.1.2 Cleaning the Nucleofection® Vessel Tray (X, Y, or 96-well Unit)
To allow convenient cleaning, the Nucleofection® Vessel tray can be
removed from the X, Y, and 96-well Unit. To dismount the tray please
follow the instructions below:
•
•
•
•
•
Figure 2.42: System settings screen
•
•
•
•
Open the “Settings” menu after vessel type selection
Press “Cleaning Mode”.
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 “Finish”.
The drawer will close and the tray will be moved to its home position.
4D-Nucleofector® Hardware Manual | 31
2
2.13.2 Left menu
Activate the left menu by tapping on the accordion icon on the left side
of the screen (Figure 2.4).
2.13.2.2 Results
This menu will display a list of result files and allows you to manage
them. Result files are listed in chronological order with latest results at
the top.
Figure 2.43: Accordion menu drop down.
Tapping on home will close this screen and returns to the home screen
(Figure 2.7).
2.13.2.1 E xperiments
This menu displays a list of experiments and allows you to view
additional information by pressing the “View” field. To load a predefined
experiment from a USB stick, insert the stick into the USB port on
the front of the Core Unit, press “Import ” and follow the instructions
displayed on the screen (Figure 2.44, B).
A
Figure 2.45: Result screen. All saved results are displayed. Scroll the list to find the result or use the arrows on the right. Tapping on “View” will display the experiment result.
To open existing result files just select the file you would like to open
and press “View”. 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 “Export”
(Figure 2.45).
2.13.2.3 Custom Cell Type Codes
The software offers the option to add custom Cell Type Code for
choosing a defined combination of a Nucleofector® Program and
Solution, e.g., after an optimization. Custom Cell Type Codes are included
in alphabetical order
To create a new custom Cell Type Code or to edit an existing Cell Type
Code:
B
Figure 2.44: Experiment Screen. (A) all available experiments are listed with information on the available cuvette format. Check the box and tap on “Export” to export the
experiment file or tap on “Load” to start the experiment. Tap on “View” to see details
about the experiment. (B) tap on “Import” to import an experiment from an USB Stick
and follow the instructions.
32 | 4D-Nucleofector® Hardware Manual
• Press “New” or select an existing program and press “Edit”.
• A screen will open allowing to select “Pulse Code” and “Solution” field
and a “Unit” selection (Figure 2.46, A and B).
A
2.13.2 Lonza programs
The “Cell Type Code” list can be exchanged independently from a
firmware update (see chapter 2.13.4). For uploading a new “Cell Type
Code” list follow instructions below:
• Insert a USB stick comprising the file you want to upload (*.pd) at
the front or the rear of the Nucleofector® Device.
The bell symbol at the top of the screen shows a notification (red dot).
Tapping on the bell icon shows further information.
A
B
B
Figure 2.46: (A) Typing the name of the custom Cell Type Code. (B) Overview of conditions for custom Cell Type Code. Press “Save” to save the conditions, press “Clear” to
start again.
• Define a name for your custom program by typing, e.g., the cell
name.
• 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 “Apply”.
• 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.
• Select functional units for which the code should apply (either X, 96well and LV Unit, or Y Unit).
• Confirm the setting for your custom program by pressing “Save”.
• The custom program can now be selected for an experiment by
pressing the “Cell Type Code” field during experiment setup (Figure
2.15).
Custom program codes will not be overwritten in case an updated
program definition file is installed (see chapter 2.13.3).
Figure 2.47: (A) Message upon insertion of USB stick with respective program file.
(B) message after tapping on the program update button.
• Choose “Program updates available” by tapping in the field.
• Tap on “Update”
• The new program files will be updated and the Nucleofector® Core
Unit will restart.
2.13.4 Firmware update
Due to technical improvement of the operation software, the
4D-Nucleofector® System may require an update of its firmware. To
update the firmware proceed as follows:
• Start the 4D-Nucleofector® System.
• The home screen will appear.
• Plug in the USB stick containing the new firmware (as provided by
Lonza)
• The bell symbol at the top of the screen shows a notification (red
dot). Tapping on the bell icon shows further information.
• Follow the instructions on the screen to update the firmware. The
Update will be loaded and the Nucleofector® Core Unit will restart.
NOTE: More comprehensive firmware updates may not be possible via
USB upload and require return of the system to Lonza.
4D-Nucleofector® Hardware Manual | 33
2
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.
Issue
Possible error
Solution
Low survival rate
Cells were kept in Nucleofector® Solution too long
Transfer cells immediately into pre-warmed medium as recommended in the
optimized protocol.
Cells were damaged by harvesting procedure or through handling
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 culture conditions were suboptimal
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®
procedure. For further details please refer to the dedicated optimized protocol.
Multiple use of cuvettes
It is strongly recommended to use the Nucleofection® Vessels only once, because
the high voltage pulses that are applied drastically affect their physical integrity.
Poor DNA quality
DNA used for Nucleofection® Experiments should be of high purity. We strongly
recommend endotoxin-free preparation of the DNA. Do not use procedures involving
phenol /chloroform treatment.
DNA amount is too low
Depending on cell type and Nucleofection® Vessel a certain DNA amount per sample
is recommended (for details please refer to respective optimized protocol). If both,
transfer efficiency and cell mortality are low, the DNA amount could be increased.
Increasing DNA amount may lead to higher transfection efficiency, but at the same
time result in higher cell mortality.
Cell number in Nucleofection® Sample too high or too low
Please use the cell numbers recommended in the dedicated optimized protocol.
Poor DNA quality
DNA used for Nucleofection® Experiments should be of high purity. We strongly
recommend endotoxin-free preparation of the DNA. Do not use procedures involving
phenol /chloroform treatment.
Low efficiency
If the problem cannot be resolved, please call Lonza’s Scientific Support
Team
Europe
Phone: + 49 221 99199 400
E-mail: [email protected]
North America
Phone: 800 521 0390 (toll free)
E-mail: [email protected]
34 | 4D-Nucleofector® Hardware Manual
3.2
Rescue of samples (X and Y Unit)
C
D
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 9) the process differs
slightly (Figure 3.1).
Model variant A (Older):
• Unplug system from main power
• Remove side panel from the X or Y Unit (Figure 3.1, A)
• Turn wheel into direction of the red arrow as far as it will go
(Figure 3.1, B)
B
A
Figure 3.1: (C/D) Rescue of samples (continued)
E
Unlocked
Figure 3.1: (A/B) Rescue of samples
Model variant B (Newer) and C (Next generation 4D-Nucleofector®
System):
• Unplug system from main power
• Remove the terminator cap or cable connection to next unit (Figure
3.1, C/E)
• Insert screw driver supplied with the 4D-Nucleofector® System in the
lower hole of the plug receptacle (Figure 3.1, D for Model variant B,
figure 3.1 F for model variant C )
• Turn the screw manually counter-clockwise until the screw blocks
(may require > 60 turns). Turn back clockwise one full turn.
F
Figure 3.1: (E/F) Rescue of samples (continued)
Further steps for all model variants (A, B, and C):
•
•
•
•
G
Open the front flap manually (Figure 3.1, G)
Pull the tray cautiously towards you (Figure 3.1, H)
Remove the Nucleocuvette® Vessels and rescue your cells.
Contact your Lonza representative to solve potential technical issue.
H
Figure 3.1: (G/H) Rescue of samples (continued)
4D-Nucleofector® Hardware Manual | 35
3
4 Error codes of the
4D-Nucleofector System
®
4.1 Color coding of wells
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.
Table 4.1: Color code of wells
Parameter File
Result File, OK
Result File, Warning
Result File, Error
Skipped Well
Undefined
Sample
No Program
No DNA
For 4D-Nucleofector® LV Unit experiments no well coding exists because
a Nucleocuvette® Cartridge consists of several segments. Symbols
are used to provide a summary rating for the whole sample based
on the percentage of segments which were pulsed properly (OK)
and segments where an error occurred. Following thresholds were
empirically defined by Lonza:
4.2 Error codes
The most recent table with error codes, code explanations and the
recommended handling procedures can be viewed at:
www.lonza.com/nucleofection-error-codes
If a problem cannot be resolved, please call Lonza’s Scientific Support
Team.
Europe
Phone: + 49 221 99199 400
E-mail: [email protected]
North America
Phone: 800 521 0390 (toll free)
E-mail: [email protected]
36 | 4D-Nucleofector® Hardware Manual
4.3
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. Such events are not considered as an error
and just indicated as a notes. 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 content, 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.
Do not re-use consumables.
5 Purchaser notification
Purchaser notification
The purchase of the 4D-Nucleofector® System (the “Purchased Device”)
conveys to the buyer a limited non-transferable, non-exclusive license
(the “License”) for the use of the Purchased Device and Lonza’s
proprietary Nucleofector® Process and methods (the “Licensed
Process”) without the right to sublicense.
license to the Purchased Device or the Licensed Process for purposes
other than research, contact our Licensing Department, at +49 221
99199 0 or e-mail: [email protected]
Representations and warranties
The License is limited to research use only. The License does not include
the right to use the Purchased Device, its components or materials, or
the Licensed Process for any Commercial Purpose, as defined below.
Notwithstanding the foregoing, the License permits the buyer to use the
Purchased Device in combination with the Licensed Process to perform
services internally for affiliates, subsidiaries, sections, departments
or scientific collaborators, pro- vided that such services are not for any
Commercial Purpose.
Lonza represents and warrants that, it is the holder of intellectual
property, including patents, patent applications, copyrights, trademarks
and technical and scientific knowledge with respect to the Purchased
Device and the Licensed Process and that, to its knowledge, it has the
right to grant the License provided herein. Lonza further represents
and warrants that it has no knowledge of any rights of third parties
which supersede its intellectual property rights to the Purchased Device
and the Licensed Process and that Lonza has full corporate power and
authority to grant the License provided hereunder.
Under no circumstances shall the Purchased Device or the Licensed
Process be used for testing or treatment in humans. The buyer,
through the purchase of the Purchased Device, acknowledges that
the Purchased Device and Licensed Process are not diagnostic or
therapeutic tools and shall not be used as an accessory or complement
to such tools.
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.
“Commercial Purposes”, as used herein, means the sale, license,
lease, export, transfer or other distribution of the Purchased Device,
its components or materials, or the Licensed Process to a person
or entity other than the buyer for financial gain or other commercial
purposes and/or the use of the Purchased Device, its components or
materials, or the Licensed Process: (1) in manufacturing or producing
a product for general sale or products for use in the manufacture
of products ultimately intended for general sale; (2) to provide a
service, information, or data to a person or entity other than the buyer
for financial gain; or (3) for therapeutic, diagnostic or prophylactic
purposes.
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.
The License does not permit the buyer to manufacture, copy, reproduce,
transmit, distribute, sell, lease, transfer or sublicense the Licensed
Process and/or the Purchased Device. For information on obtaining a
THE EXPRESS WARRANTY PROVIDED ABOVE IS LONZA’S ONLY WARRANTY
RELATING TO THE PURCHASED DEVICE OR THE LICENSED PROCESS
AND IS IN PLACE OF ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED,
STATUTORY OR OTHERWISE, INCLUDING WARRANTIES AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, ALL OF WHICH ARE
EXPRESSLY EXCLUDED. BUYER AGREES THAT LONZA HAS MADE NO
UNDERTAKINGS, AGREEMENTS OR REPRESENTA- TIONS, EXPRESSED OR
IMPLIED WITH RESPECT TO THE PURCHASED DEVICE OR THE LICENSED
PROCESS, OTHER THAN THOSE EXPRESSLY SET FORTH HEREIN.
4D-Nucleofector® Hardware Manual | 37
4
5
NOTWITHSTANDING ANYTHING TO THE CONTRARY SET FORTH HEREIN,
IN NO EVENT WILL LONZA BE LIABLE FOR LOST PROFITS, DAMAGE
TO REPUTATION, OR LOSS OF DATA, OR FOR ANY SPECIAL, INDIRECT,
INCIDENTAL, PUNITIVE, OR CONSEQUENTIAL DAMAGES, HOWEVER CAUSED,
ON ANY THEORY OF LIABILITY AND WHETHER OR NOT LONZA HAS BEEN
ADVISED OF THE POSSIBIL- ITY OF SUCH DAMAGES, ARISING UNDER ANY
CAUSE OF ACTION AND ARISING IN ANY WAY OUT OF BUYER’S USE OF
THE PURCHASED DEVICE OR THE LICENSED PROCESS. BUYER HEREBY
AGREES THAT TO THE FULLEST EXTENT PERMITTED BY LAW, LONZA’S
LIABILITY TO BUYER FOR ANY AND ALL INJURIES, CLAIMS, LOSSES,
EXPENSES, OR DAMAGES WHATSOEVER, ARISING UNDER ANY CAUSE OF
ACTION AND ARISING IN ANY WAY OUT OF BUYER’S PURCHASE OR USE OF
THE PURCHASED DEVICE OR LICENSED PROCESS SHALL BE LIMITED IN
THE AGGREGATE, IN LONZA’S SOLE AND ABSOLUTE DISCRETION, TO REPAIR,
REPLACEMENT, OR REFUND OF THE EQUIPMENT OR EQUIPMENT PART,
AS APPLICABLE, OR THAT PORTION OF THE PURCHASE PRICE APPLICABLE
TO THE EQUIPMENT OR THE EQUIPMENT PART, AS APPLICABLE,
WHICH VIOLATES AN EXPRESS WARRANTY PROVIDED HEREUNDER.
NOTWITHSTANDING ANYTHING HEREIN TO THE CONTRARY, THE LIABILITY
OF LONZA WITH RESPECT TO A CLAIM OF ANY KIND WHATSOEVER, SHALL
NOT EXCEED THE INVOICE PRICE OF THE PARTS, REPLACEMENT PARTS OR
SERVICE AS TO WHICH THE CLAIM IS MADE.
38 | 4D-Nucleofector® Hardware Manual
Buyer assumes all responsibility and liability for injury or damages
resulting from its handling, possession, use or sale of the Purchased
Device or Licensed Process including, but not limited to any injury or
damage resulting from the use of the Purchased Device or Licensed
Process in Buyer’s operations or in combination with other substances,
materials or products. Buyer agrees to defend, indemnify and hold
Lonza and its affiliates and their respective directors, officers,
employees, agents harmless from and against all claims, losses,
damages, liabilities and expenses (including attorneys’ fees and other
litigation or settlement costs) arising out of (a) Buyer’s handling,
possession, use or sale of the Purchased Device or Licensed Process,
(b) any material breach of any Agreement between Lonza and Buyer
or other terms and conditions relating to the use of the Purchased
Device or Licensed Process provided to Buyer by Lonza, or (c) the
gross negligence or willful misconduct of Buyer or any of its affiliates in
performing any activity relating to the subject matter hereof.
Lonza does not assume any liability that the Licensed Process,
the Purchased Device or any product resulting from the use of the
Licensed Process or use of the Purchased Device shall be approved by
governmental authorities or any other institution or supervisory board
or similar agencies.
6 Technical specifications
Technische Daten, Specifications, Spécification
Spannung / power / électrique
100–240 VAC
Frequenz / frequency / fréquence
50–60 Hz
Stromverbrauch / consumption / consommation
0 VA (power off) / 20 VA (idle) / 140 VA
(busy - during transfection)
Sicherung / fuse / fusible
T 2.5A, L250V
Betriebsumgebung / operating temperature /
environment
+15°C to +40°C, (59°F - 104°F) non-condensing
Transfektionen pro Minute / transfections per
minute / transfections par minute
Nucleocuvette® Strips)
X Unit:
16-32 reactions
20-26 reactions
using 16-well Nucleocuvette® Strips
using 100 µL Nucleocuvette® Vessels
Y Unit:
8-10 reactions
using Dipping Electrodes
LV Unit:
2-3 mL
using LV Nucleocuvette® Cartridges
96-well Unit:
32-44 reactions
using 96-well Nucleocuvette® Plate
Schnittstellen / interfaces / interface
USB network connection
Spannungsbegrenzung / voltage limitation /
limitation de tension
1500 V
Strombegrenzung / current limitation /
limitation de courant
100 A
Klassifikation/ classification/ classification
IP 20 - protection against foreign materials
Splash-proof until 15° inclination
Group 1, Class B
Abmessungen / dimensions / dimensions
(w × h × d)
Gewicht / weight / poids
Core Unit:
X Unit:
Y Unit:
LV Unit:
96-well Unit:
Hersteller / supplier / manufacture
Lonza Cologne GmbH
Nattermannallee 1
D-50829 Cologne, Germany
Herstellungsdatum / date of manufacture /
date de production number
The manufacturing year is encoded by the second and third digit
of the serial number, e.g., serial number x16xxxxx was manufactured in 2016.
4.4 kg (9.7 lb)
4.7 kg (10.4 lb)
5.0 kg (11.0 lb)
5.7 kg (12.5 lb)
4.7 kg (10.4 lb)
6
4D-Nucleofector® Hardware Manual | 39
Contact us
North America
Customer Service: +1 800 638 8174 (toll free)
Fax: +1 301 845 8338
[email protected]
Scientific Support: +1 800 521 0390 (toll free)
[email protected]
Europe
Customer Service: +49 221 99199 400
[email protected]
Scientific Support: +49 221 99199 400
[email protected]
International
Contact your local Lonza distributor
Customer Service: +1 301 898 7025
[email protected]
International Offices
Australia
Belgium
Brazil
China
France
Germany
India
Japan
Luxemburg
Singapore
The Netherlands
United Kingdom
+61 3 9550 0883
+32 87 321 611
+55 11 4028 8000
+86 21 6305 8866
0800 91 19 81 (toll free)
0800 182 52 87 (toll free)
+91 124 6052941
+81 3 6264 0660
+32 87 321 611
+65 6521 4379
0800 022 4525 (toll free)
0808 234 97 88 (toll free)
Lonza Cologne GmbH – 50829 Cologne Germany
For research use only. Not for use in diagnostic procedures.
The Nucleofector® Technology is covered by patent and/or patent pending rights owned by the Lonza Group Ltd or its affiliates.
The Nucleofector® Technology, comprising Nucleofection® Process, Nucleofector® Device, Nucleofector® Solutions, Nucleofector® 96-well
Shuttle® System and Nucleocuvette® Plates and Modules is covered by patent and/or patent pending rights owned by Lonza Cologne GmbH.
Nucleofector, Nucleofection, Nucleocuvette, maxGFP and 96-well Shuttle are trademarks of Lonza Cologne GmbH in Germany and / or the
U.S. and / or other countries.
All trademarks belong to Lonza, registered in USA, EU or CH or to third party owners and used only for informational purposes. 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. Any user
must make his own determination and satisfy himself that the products supplied by Lonza Group Ltd or its affiliates and the information
and recommendations given by Lonza Group Ltd or its affiliates are (i) suitable for intended process or purpose, (ii) in compliance with
environmental, health and safety regulations, and (iii) will not infringe any third party’s intellectual property rights. The user bears the
sole responsibility for determining the existence of any such third party rights, as well as obtaining any necessary licenses. For more
details: www.lonza.com/legal.
©2021 Lonza. All rights reserved.
lonza.com/transfection
bioscience.lonza.com
Cultek S.L.U.
Your partner in life sciences
CD-MN050 05/21
917 290 333
[email protected]
www.cultek.com

advertisement

Related manuals

Download PDF

advertisement