Life technologies invitrogen Neon User manual

User Manual
Neon™Transfection System
For transfecting mammalian cells, including primary and
stem cells, with high transfection efficiency
Catalog no. MPK5000
Rev. Date: 1 April 2010
Manual Part no. 25-1055
MAN0001557

ii

Contents
Product Contents......................................................................................................................................... v
Unpacking the Neon™Transfection System ........................................................................................... vi
Neon™Transfection System ..................................................................................................................... vii
Introduction ...................................................................................................................1
About the Product........................................................................................................................................1
Description of Parts......................................................................................................................................3
Methods .........................................................................................................................6
Getting Started..............................................................................................................................................6
General Guidelines.....................................................................................................................................12
Using the Neon™Transfection System ....................................................................................................14
Optimization Protocol for DNA and siRNA ..........................................................................................22
Troubleshooting..........................................................................................................................................31
Neon™Device Error Messages..................................................................................................................33
Appendix......................................................................................................................34
Repackaging the Instrument.....................................................................................................................34
Product Specifications................................................................................................................................35
Safety Information......................................................................................................................................36
Accessory Products ....................................................................................................................................38
Technical Support.......................................................................................................................................39
Purchaser Notification...............................................................................................................................40
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iv

Product Contents
Upon Receiving
the Device Examine the unit carefully for any damage incurred during transit. Any damage
claims must be filed with the carrier. The warranty does not cover in-transit
damage. To register the device, activate your warranty, and be notified of important
updates, go to www/invitrogen.com/neon.
Neon™
Transfection
System
The contents of the Neon™Transfection Systems are listed below. The Neon™
Transfection System is shipped at room temperature.
See page 3for specifications and description of the Neon™Transfection System,
and page 6to set up the device.
Product Quantity
Neon™Transfection Device 1
Specific Power Cord
(for US/Canada/Taiwan/Japan, Europe, and UK) 4
Neon™Pipette 1
Neon™ Pipette Station 1
Instruction Manual 1
Neon™Kits The Neon™Kits are used with the Neon™Transfection Systems for efficient
transfection of mammalian cells and are available separately from Invitrogen
(page 38). The kits are available in two formats for electroporation of 10 μL and
100 μL samples.
The following components are included with the Neon™Kit. The Neon™Kits are
shipped at room temperature.
Upon receipt, store the kit at room temperature. After use, store buffers at 4ºC
and all remaining kit components at room temperature.
Neon™ Kit, 10 μL Neon™ Kit, 100 μLItem
Cat. no. MPK1025
(50 reactions)
Cat. no. MPK1096
(192 reactions)
Cat. no. MPK10025
(50 reactions)
Cat. no. MPK10096
(192 reactions)
Neon™Tips 25 tips (10 μL) 96 tips (10 μL) 25 tips (100 μL) 96 tips (100 μL)
Neon™Tubes 5 20 5 20
Resuspension Buffer R
(Proprietary)
1 mL 3 × 1 mL 10 mL 30 mL
Resuspension Buffer T
(Proprietary)
1 mL 3 × 1 mL 10 mL 30 mL
Electrolytic Buffer E
(Proprietary)
75 mL 2 × 150 mL Not applicable Not applicable
Electrolytic Buffer E2
(Proprietary)
Not applicable Not applicable 75 mL 2 × 150 mL
Intended Use For research use only. Not intended for any animal or human therapeutic or
diagnostic use.
v

Unpacking the Neon™Transfection System
Unpacking
Instructions Follow the instructions below to unpack the Neon™Transfection System. The
weight of the Neon™device is 13.2 pounds (6 kg).
1. Cut plastic tapes and remove the outer box. Save the outer box and other
packaging material (in case you need to transport or ship the unit).
2. Remove the plastic bag from the top containing the manual, the Neon™
Pipette box containing the pipette, and then remove the plastic bag
containing the power cords from the box.
3. Remove the Neon™device and Neon™pipette station from the box and place
on a flat, level surface.
4. Set up the Neon™Transfection System as described on page 6.
vi

Neon™Transfection System
Front View The front view of the Neon™device is shown below.
Touchscreen
Rear View The rear view showing various parts of the Neon™device are shown below.
The USB port (need to unscrew the panel to view the port) is used to connect a
USB memory drive. The AC inlet is to connect to the power outlet on the wall,
and high voltage and sensor port is to connect the high voltage and sensor
connector of the Neon™Pipette Station to the unit.
Sensor
port AC inlet
Continued on next page
High voltage
port Power switch
USB port panel
Fan
vii

Neon™Transfection System, Continued
Neon™Pipette
Station The Neon™Pipette Station is supplied with a high voltage and sensor connector
which connects the pipette station to the Neon™device. The Neon™Pipette with a
Neon™Tip and Neon™Tube is then used with the Neon™Pipette Station for
electroporation of mammalian cells. The Neon™Pipette Station contains two
electrodes.
Connector
User Interface The touchscreen user interface of the Neon™device consists of:
The touchscreen buttons to operate the device
The Digital Display that shows the protocol that is in use and various
parameters of the protocol.
Digital
display
Touchscreen
buttons
viii

Introduction
About the Product
Neon™
Transfection
System
The Neon™Transfection System is a novel, benchtop electroporation device
that employs an electroporation technology by using the pipette tip as an
electroporation chamber to efficiently transfect mammalian cells including
primary and immortalized hematopoietic cells, stem cells, and primary cells.
The Neon™Transfection System efficiently delivers nucleic acids, proteins, and
siRNA into all mammalian cell types including primary and stem cells with a
high cell survival rate. The transfection is performed using as few as 1 × 104or
as many as 5 × 106cells per reaction using a sample volume of 10 μL or 100 μL
in a variety of cell culture formats (60 mm, 6-well, 48-well, and 24-well).
The Neon™Transfection System uses a single transfection kit (Neon™Kit) that
is compatible with various mammalian cell types including primary and stem
cells thereby avoiding the need to determine an optimal buffer for each cell
type.
The Neon™Transfection System offers open and transparent protocols that are
optimized for ease of use and simplicity. The Neon™device is preprogrammed
with one 24-well optimization protocol to optimize conditions for your nucleic
acid/siRNA and cell type, or you can program and store up to 50 cell-specific
protocols in the Neon™device database. Optimized protocols for many
commonly used cell types are also available on www.invitrogen.com/neon for
your convenience to maximize transfection efficiencies for your cell types.
See page 3for details on various parts of the system.
System
Components The Neon™Transfection System consists of:
Neon™Device
The Neon™device is a simple, user friendly benchtop electroporation
device that employs the pipette tip as an electroporation chamber to
efficiently transfect mammalian cells including primary and immortalized
hematopoietic cells, stem cells, and primary cells. The device is
preprogrammed with a 24-well optimization protocol and supports a
database to store up to 50 user-specified protocols. See page 3for details.
Neon™Pipette Station
The Neon™Pipette Station is a unique component of the system and holds
the Neon™Pipette during electroporation and protects the user from any
electrical shock exposures. The Neon™Tube which has an electrode near
the bottom is inserted into the pipette station to transfer the electric field
from the electrode inside the Neon™ Tip. See page 3for details.
Neon™Kits (not supplied with the device)
The Neon™Kits contain the Neon™Tips, Neon™Tubes, and buffers for
electroporation. The Neon™Kits are available in two formats for
electroporation of 10 μL or 100 μL samples (page 38 for ordering
information). See page 3for details on Neon™Tips and Tubes.
Continued on next page
1

About the Product, Continued
System Overview Unlike standard cuvette based electroporation, the Neon™Transfection System
uses a unique electroporation reaction chamber, the Neon™Tip that delivers a
high electric field to the biological sample. The Neon™ Tip maximizes the gap
size between the two electrodes while minimizing the surface area of each
electrode. As a result, the sample experiences a more uniform electric field,
minimal pH change, less ion formation, and negligible heat generation.
This next generation electroporation technology overcomes many of the
limitations associated with standard cuvette based electroporation thereby
increasing transfection efficiency and cell viability, and providing an ergonomic
workflow.
VVV
The transfection occurs in the uniquely designed Neon™Tip using simple 3-
step procedure.
1. Load a mixture of harvested cells and molecules to be delivered (e.g., DNA,
RNA, siRNA) into the Neon™Tip.
2. Plug the Neon™Pipette with Neon™Tip into position in the Neon™Pipette
Station with Neon™Tube; select your protocol on the device, and press
Start.
3. Unplug the Neon™Pipette and transfer your transfected cells into a tissue
culture vessel containing the appropriate medium.
Features Important features of the Neon™Transfection System are listed below:
User-friendly Neon™device benchtop design that easily fits in your tissue
culture hood for easy, efficient transfection of a wide variety of
mammalian cells including primary and stem cells
Ability to transfect 1 × 104–5 × 106cells per reaction in a sample volume of
10 μL or 100 μL in a variety of cell culture formats (60 mm, 6-well, 48-well,
and 24-well)
Utilizes a single buffer system for all cell types except primary suspension
blood cells
Simple touch screen interface for easy programming of electroporation
parameters
Available with one pre-programmed 24-well optimization protocol and
the option to customize up to 50 cell specific protocols
Built-in safety features in the device to enhance user safety
Continued on next page
2

Description of Parts
Neon™Device The Neon™device is a simple,
user friendly benchtop
electroporation device. When
used with a Neon™Pipette
Station and Neon™Kits, the
Neon™device efficiently
transfects mammalian cells
including primary and stem
cells. The device is
preprogrammed with a 24-well
optimization protocol and
supports a database to store up
to 50 user-specified protocols.
See page vii for a front and
rear view of the device.
Neon™Pipette The Neon™Pipette utilizes a positive
displacement pipette mechanism for pipetting
mixtures containing cells and nucleic acid or
siRNA. The Neon™Pipette is a fixed volume
pipette and permanently calibrated at the
manufacturing stage and does not require any
further calibration.
The Neon™Pipette is designed for use with
Neon™Tips only. Do not use any other tips with
the Neon™Pipette.
Continued on next page
3

Description of Parts, Continued
Neon™Pipette
Station The Neon™Pipette Station is a unique component of the Neon™Transfection
system. It holds a Neon™Pipette during electroporation procedures. The
Neon™Pipette Station is equipped with many safety sensors and protection
mechanisms that protects the user from any exposures to an electrical shock.
The Neon™Pipette Station is connected to the Neon™device using the high
voltage and sensor connector (see page 6for details).
The Neon™Pipette Station also holds the Neon™Tube which has an electrode
near the bottom that transfers the electric field from the electrode inside the
Neon™ Tip.
Area to insert
Neon™Tube
Neon
™
Pipette
Station Connector
Neon™Tube The Neon™Tube holds the Electrolytic Buffer during electroporation and is
inserted into the Neon™Pipette Station. The Neon™Pipette with the Neon™Tip is
then inserted into the Neon™Tube which has an electrode near the bottom that
transfers the electric field from the electrode inside the Neon™ Tip. The Neon™
Tubes are supplied with Neon™Kits as well as available separately (page 38).
To avoid contamination, we strongly recommend using the tubes for a
maximum of 10 times only. We recommend changing tube and buffer when
switching to a different plasmid DNA/siRNA or cell type.
Tube Specifications:
Material: Polystyrene
Capacity: 2.5–4 mL
Buffer
Electrode
Continued on next page
4

Description of Parts, Continued
Neon™Tips The Neon™Tips are disposable tips composed of a tip and piston used with the
Neon™Pipette. The Neon™Tips contain a gold-plated electrode to create a
disposable electric chamber for the delivery of a high electric field to biological
samples. The Neon™Tips are supplied with Neon™Kits in two formats to
support operating volumes of 10 μL and 100 μL, respectively (page 38 for
ordering information).
To ensure repeatability and eliminate variation of the transfection conditions
within or between experiments, we recommend that you do not use the
Neon™Tip for more than 2 times. Oxide formation at the piston surface area
can be generated if the tips are used more than 2 times, which decreases
electrode function of the piston.
Tip Specifications:
Material: Polypropylene
Capacity: 10 μL or 100 μL
Piston
Mounting
stem
Gold electrode
Tip
5

Methods
Getting Started
Installing the
Neon™Device with
Pipette Station
1. Unpack the Neon™device as instructed on page vi.
2. Four power cords are shipped with the device to ensure that the cord you use
is compatible with your local socket format.
3. Place the Neon™device on a level laboratory bench. Keep the area around the
unit clear to ensure proper ventilation of the unit.
Note: The Neon™device has a small footprint and can be easily set up in the tissue
culture hood for convenience.
4. For your safety: Position the device properly such that the power switch and
AC inlet located on the rear of the unit (page vii) are easily accessible. Be sure
to position the device such that it is easy to disconnect the unit.
Note: Since Neon™device is air-cooled, its surface may become hot during operation.
When installing the device, leave a space of more than 10 cm from the back of the device.
5. Place the Neon™Pipette Station near the Neon™device.
6. Connect the high voltage and sensor connector on the Neon™Pipette Station to
high voltage port and sensor port on the rear side of Neon™device,
respectively.
Be sure to align the ridge indicated by a white arrow on the sensor connector
on the Neon™Pipette Station with a groove indicated by a white dot on the
sensor port of the Neon™device (see figure below for details).
Important! To connect or disconnect the sensor connector to the Neon™device,
always handle the sensor connector using the cord plug and not the cord cable.
7. Ensure the AC power switch is in the Off position (page vii).
Continued on next page
6

Getting Started, Continued
Installing the
Neon™Device with
Pipette Station,
Continued
8. Attach the power cord to the AC inlet on the rear of the Neon™device and
then to the electrical outlet. Use only properly grounded AC outlets and power
cords.
9. To turn on the power, press the main power switch on the rear of the unit to
ON position. The digital display shows start up screen (next page).
10. The Neon™device is operated by the touch screen on the front of the device.
You can easily input electroporation parameters by lightly touching the touch
screen with a finger tip or a touch screen pen. See next page for details.
You are ready to use the Neon™Transfection System. See page 14 for details.
Registering the
Device Visit www.invitrogen.com/neon to register the device and activate your
warranty or extended warranty, and ensure that you receive product updates,
special offers, and faster service.
Electroporation
Protocol Options There are three options to select an electroporation protocol for your cell type:
If you already have the electroporation parameters for your cell type, input
the parameters in the Input Window (see below).
If you wish to add cell-specific electroporation parameters to the database
on the device for future use, input the parameters in the Database Window
(page 9). You can also view our library of protocols for commonly used cell
types from www.invitrogen.com/neon and in put the parameters in the
Database Window (see below) for various cell types.
If you do not have any specific electroporation parameters for your cell type
and wish to perform optimization, use the Optimization Window
(page 11).
Input Values Limit The Neon™device is designed to only input certain values and limits for each
value are listed below. If your input value exceeds the maximum value, an
error is displayed.
Input Voltage range: 500–2,500 V
Input Pulse Width range: 1–100 ms
Input Pulse Number range: 1–10
Continued on next page
7

Getting Started, Continued
Input Window,
Continued To create a cell specific protocol, if you already have the electroporation
parameters for your cell type:
1. Press the power switch (located on the rear side of the unit, page vii) to turn
ON the Neon™device. The unit checks to ensure that the Neon™Pipette
Station is connected to the device and then the start up screen is displayed.
2. Press Voltage to activate the number key pad to input voltage value. Press
the desired voltage value and press Done to save the value.
Note: If any input value is out of the limit, an error message is displayed and the
lowest value of limit is automatically set.
3. Press Width to activate the number key pad to input width value. Press the
desired width value and press Done to save the value.
4. Press Pulses to activate the number key pad to input pulse value. Press the
desired pulse value and press Done to save the value.
5. If you wish to save these electroporation parameters, press Save on the
main screen to save the protocol in the database.
6. Press the desired protocol number button to edit the protocol. The selected
protocol is highlighted.
7. Once the Edit screen is displayed, enter the User name by pressing the key
pad buttons. The cursor automatically moves to the next field Protocol and
is highlighted red.
Continue to enter the information for Voltage, Width, and Pulse.
8. Press Enter to save the information in the database.
9. Proceed to preparing cells (pages 16–17) and DNA, and setting up the
Neon™Pipette Station for electroporation (page 14).
Continued on next page
8

Getting Started, Continued
Database Window Enter cell-specific protocols into the database. The database can store up to
50 cell-specific protocols.
1. Press the power switch (located on the rear side of the unit, page vii) to
turn ON the Neon™device. The unit checks to ensure that the Neon™
Pipette Station is connected to the device and then the start up screen is
displayed.
2. Press Database button to start the database window. To scroll through the
protocols in the database, use the right/left scroll buttons near the
Database button.
The Database window shows:
Number button: Indicates protocol number
User and Protocol: Displays the user and protocol name
Parameters (Voltage, Width, Pulse): Displays the electroporation
parameter for each protocol
Function buttons (Load, Edit, and Delete): Used to load, edit, or delete
a protocol. The function buttons are activated only after a protocol is
selected.
Page scroll: To scroll to next or previous page
3. Press the desired protocol number button to edit the protocol. The selected
protocol is highlighted.
Continued on next page
9

Getting Started, Continued
Database Window,
Continued
4. Once the Edit screen is displayed, enter the User name by pressing the key
pad buttons. The cursor automatically moves to the next field Protocol
and is highlighted red.
Continue to enter the information for Voltage, Width, and Pulse.
If you wish to password protect the protocol, enter the Password (up to
7 characters) and Repeat Password information using the key pad.
5. Press Enter to save the information in the database. To exit the edit screen
without saving the parameters, press X.
6. The database window is displayed. Press the desired protocol and then
press Load to load electroporation parameters from the database.
7. Proceed to preparing cells (pages 16–17) and DNA, and setting up the
Neon™Pipette Station for electroporation (page 14).
8. To delete a protocol from the database, select the protocol by pressing the
protocol number button. Press Delete. If the protocol in the database was
password protected, a password screen is displayed. Enter the password
and press Enter to delete the protocol.
Continued on next page
10

Getting Started, Continued
Optimization
Window Perform optimization of electroporation parameters using the preprogrammed
24-well optimization protocol. These protocols are locked and cannot be
edited.
1. Press the power switch (located on the rear side of the unit, page vii) to
turn ON the Neon™device. The unit checks to ensure that the Neon™
Pipette Station is connected to the device and then the start up screen is
displayed.
2. Press Optimization button to start the optimization window. To scroll
through the protocols, use the right/left scroll buttons near the
Optimization button.
The Optimization window shows:
Number button: Indicates protocol number
User and Protocol: Displays the optimization and well number
Parameters (Voltage, Width, Pulse): Displays the electroporation
parameter for each protocol
Load Function buttons: Used to load a protocol. The Load button is
activated only after a protocol is selected.
Page scroll: To scroll to next or previous page
3. Press the desired protocol number button. The selected protocol is
highlighted. Press Load to load the protocol. To exit the screen without
loading the protocol, press X.
4. The electroporation parameters are displayed on the start up screen.
5. Proceed to preparing cells (pages 16–17) and DNA, and setting up the
Neon™Pipette Station for electroporation (page 14).
Upgrading the
Firmware Upgrades for the Neon™device firmware are available. To download Neon™
device firmware upgrades, go to www.invitrogen.com/neon. Follow
instructions on the page to download the upgrades.
11

General Guidelines
Recommended
Kits To use the Neon™device for electroporation of mammalian cells, you need to
purchase the Neon™Kits from Invitrogen. Ordering information is on page 38.
Do not use any other kits with the unit.
R
E
C
O
M
M
E
N
D
A
T
I
O
N
To obtain the best results, follow these recommendations:
Based on your initial results, you may need to optimize the electroporation
parameters for your cell type and DNA/siRNA. A preprogrammed 24-
well optimization protocol is included in the device for your convenience.
Before using the device with your samples, ensure that you are able to
insert and use the Neon™Pipette and Tip correctly into the Neon™Pipette
Station (see page 14 for details).
Wear gloves, laboratory coat, and safety glasses during electroporation.
Always use the Neon™device with Neon™Kits for electroporation of
mammalian cells.
The Neon™Transfection System is compatible for use with most
mammalian cells including primary and stem cells.
Use high quality DNA and siRNA to obtain good transfection efficiency.
Follow the guidelines on pages 16–17 for cell preparation.
Use an appropriate GFP (green fluorescent protein) construct or siRNA
control (see next page for details) to determine transfection efficiency.
Discard the Neon™Tips after 2 usages and Neon™Tubes after 10 usages as
a biological hazard. We strongly recommend changing tube and buffer
when switching to a different plasmid DNA/siRNA or cell type.
Visit www.invitrogen.com/neon for a library of electroporation protocols
for commonly used cell types.
Recommended
Buffers The Neon™Kits contain two Resuspension Buffers. Use the appropriate
Resuspension Buffer based on the cell type as below. The cell-specific Neon™
transfection protocols available on www.invitrogen.com/neon indicate the
type of Resuspension buffer for use with each cell type.
Resuspension Buffer R:
Use Resuspension Buffer R with established adherent and suspension cell
lines such as 3T3-L1, HEK293, Cos7, C2C12, HeLa, HCT-15, PC-12, MDCK,
Raw264.7, U-2OS, CEM, HL-60, K-562, Jurkat, LCL, Ramos, U-937, as well as
primary adherent cells such as neuronal cells, stem cells, hepatocytes,
HUVEC, macrophage cells, dendritic cells.
Resuspension Buffer T:
Use Resuspension Buffer T with primary blood-derived suspension cells such
as T-cells, B-cells, NK cells, PBMC, monocytes.
Continued on next page
12