Axon Axoporator 800A User manual

Axoporator 800A
SINGLE-CELL ELECTROPORATOR
Theory and Operation
Part Number 2500-0158 Rev C March 2005 Printed in USA
Copyright 2005 Axon Instruments / Molecular Devices Corp.
No part of this manual may be reproduced, stored in a retrieval system, or
transmitted, in any form or by any means, electronic, mechanical, photocopying,
microfilming, recording, or otherwise, without written permission from Molecular
Devices Corp.
QUESTIONS? See Axon's Knowledge Base: http://support.axon.com
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P LEASE READ !!!!! SAFETY
There are important safety issues that you must take into account when using this
instrument. Please carefully read the safety warnings starting on page 69 before
you use this instrument.
VERIFICATION
This instrument is extensively tested and thoroughly calibrated before leaving the
factory. Nevertheless, researchers should independently verify the basic accuracy
of the controls using resistor models of their micropipettes.
WARNING
If this equipment is used in a manner not specified by the manufacturer, the
protection provided by the equipment may be impaired.
DISCLAIMER
This equipment is not intended to be used, and should not be used, in human
experimentation or applied to humans in any way.
LICENSING NOTICE
This product is sold under license from Cellectricon AB.
Verification, Warning, Disclaimer, Licensing

Table of Contents •iii
Table of Contents
Chapter 1 Introduction.......................................................................................... 1
Chapter 2 Functional Checkout............................................................................ 5
Chapter 3 Installation............................................................................................ 9
Standard Configuration ......................................................................................... 9
Micromanipulator.............................................................................................. 9
Microscope...................................................................................................... 10
Connections..................................................................................................... 10
Optional Configurations...................................................................................... 11
Oscilloscope .................................................................................................... 11
Chapter 4 Reference Section ............................................................................... 13
Audio Monitor..................................................................................................... 13
Headstage ............................................................................................................ 14
High Voltage Precautions ............................................................................... 14
Static Precautions ............................................................................................ 14
Hot Plugging ................................................................................................... 14
Types............................................................................................................... 14
Voltage Clamp Circuit .................................................................................... 14
Noise ............................................................................................................... 16
Mounting......................................................................................................... 16
Bath Connection.............................................................................................. 16
Electroporating while Recording .................................................................... 16
Cleaning .......................................................................................................... 16
Front Panel Controls............................................................................................ 17
Front Panel Display............................................................................................. 18
Rear Panel Inputs/Outputs................................................................................... 22
Table of Contents

iv •Table of Contents
Overview of Pulses and Trains............................................................................ 25
Terminology .................................................................................................... 27
Frequency equals the inverse of the duration between the start of two
successive pulses in the train........................................................................... 27
Using Axoporator’s Pulse Generator............................................................... 27
Triggering........................................................................................................ 28
Micropipette Holder ............................................................................................ 28
Holder Design.................................................................................................. 28
Holder Use....................................................................................................... 30
Holder Maintenance ........................................................................................ 31
Adapters .......................................................................................................... 31
Model Cell........................................................................................................... 32
Chapter 5 Tutorial................................................................................................33
Guidelines for Single-Cell Electroporation ......................................................... 33
Micropipettes................................................................................................... 33
Selecting the Stimulation Parameters.............................................................. 38
Overview of Protocols for Loading Dissociated Cells in Culture ................... 40
Testing the Setup ............................................................................................. 43
Chapter 6 Troubleshooting Guide ......................................................................45
General Rule.................................................................................................... 45
Symptom: High or infinite tip resistance......................................................... 45
Symptom: Decrease in tip resistance............................................................... 45
Symptom: Positional drift ............................................................................... 46
Symptom: Contents leaking from micropipette tip ......................................... 46
Symptom: Unable to load cells........................................................................ 46
Symptom: Current or power reading is out of range...................................... 47
Symptom: Grey out of pulse frequency or width reading .............................. 47
Symptom: No R
f
value is reported ................................................................. 47
Chapter 7 Specifications ......................................................................................49
AP-1A Headstage (voltage clamp)...................................................................... 49
AP-1A-1MU (standard)................................................................................... 50
AP-1A-0.1MU (optional) ................................................................................ 50
Micropipette holder ......................................................................................... 51
Main Unit ............................................................................................................ 51
Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

Table of Contents •v
Operating modes ............................................................................................. 51
Offset Voltage ................................................................................................. 52
Pulse types....................................................................................................... 52
Pulse Generation (active mode, internal) ........................................................ 52
LCD display .................................................................................................... 53
Rear panel........................................................................................................ 54
Power requirements (using the provided power supply)................................. 56
Model Cell........................................................................................................... 56
Accessories Provided .......................................................................................... 56
References .............................................................................................................. 57
Technical Assistance.............................................................................................. 61
Warranty and Repair Service.............................................................................. 63
Declaration of Conformity.................................................................................... 67
Important Safety Information.............................................................................. 69
Index....................................................................................................................... 71
Table of Contents

Introduction •1
Chapter 1
Introduction
Electroporation is commonly used for delivering macromolecules, including DNA,
RNA, dyes, and proteins, into cells. Electroporation involves two basic
components, the permeabilization of cell membranes by application of short-
duration electric field pulses, and electrophoretic delivery of molecules through
these pores (Kinosita, 1979; Weaver, 1993; Neumann et al., 1999; Ho et al., 1996).
When cells are placed in an electric field, charged ions within the cells migrate
towards the external electrodes, resulting in a build-up of charge at the poles of the
cells adjacent to the oppositely-charged extracellular electrodes. When the induced
transmembrane potential reaches approximately 0.25 V to 1 V, the electrostatic
forces holding the lipid bilayer together break down, causing a reconfiguration of
the membrane phospholipids and creation of minute pores in the small regions of
the membrane at each pole (Neumann et al., 1999; Ho et al., 1996). Once formed,
these pores (20 nm to 120 nm in diameter) remain open even after the external
electric field is removed. Molecules from the extracellular solution move into the
cell soma through these pores. Charged molecules in the extracellular solution
migrate towards the electrode of the opposite charge. Uncharged molecules will
follow the concentration gradient. When the electric field terminates, small pores
eventually collapse and the cell membrane continuity recovers over a timescale of
tens to hundreds of milliseconds trapping the molecules that have moved across the
Introduction

2 •Introduction
membrane in the cell. If, however, the pores are too large, they will not re-seal,
leading to lysis and death of the cell.
Electroporation is potentially applicable to all cell types regardless of their origin,
stage of maturation, or preparation in which they are found. Traditionally,
electroporation has been used for transfection of dissociated cells in solution. Cells
bathed in a solution of DNA are placed in a cuvette and exposed to high-voltage
pulses delivered between two large plate electrodes in the cuvette. Recently,
electroporation has been used for the bulk transfection of cells within intact tissues,
including neurons within the spinal cord (Sakamoto et al., 1998), eye (Koshiba et
al., 2000) and brain (Haas, 2002). These techniques typically involve injecting a
solution of DNA into an enclosed space, such as the lumen, or directly into the
tissue, followed by application of high-voltage pulses between two electrodes on
either side of the tissue. Controlled application of an electric field and restricted
exposure to the molecules to be delivered allow precise targeting of electroporation
to specific cells (Teruel, 1999; Atkins, 2000; Haas, 2001).
Single-cell electroporation allows targeted delivery of molecules. A number of
technical approaches have been devised to target individual cells, including
microelectrodes (Lindqvist, 1998; Olofsson, 2003), electrolyte-filled capillary tubes
(Nolkrantz, 2001) and electronic chips (Huang, 2000). A most efficient method for
single-cell electroporation employs a glass micropipette that restricts the electric
field to a single cell (Haas, 2001; Rae, 2002). This is accomplished by placing a
solution with the molecules to be delivered into the tip of the glass micropipette
whose diameter is less than the width of the target cell (approximately 0.5 µm).
After the micropipette tip is placed near, or in contact with the target cell
membrane, voltage pulses are delivered between a chlorided silver wire within the
pipette and an external chlorided ground electrode. The voltage pulses cause pores
to form in the membrane of the adjacent cell and electrophoretically drive charged
molecules from the pipette, through the pores into the cell. After pulse termination
the pipette is removed, leaving a single cell loaded with the desired molecules.
This technique leaves any surrounding cells in culture or in the intact tissue
unaltered. Single-cell electroporation can be used to deliver molecules to
individual cells within dissociated cultures, organotypic tissue cultures, acute tissue
Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

Introduction •3
slices, or in vivo. The ability to target individual cells is a great advantage for
experiments designed to test cell-autonomous effects of manipulation. Single-cell
electroporation is especially useful for studies requiring single cell resolution, such
as imaging studies of cell morphology (Haas, 2001; Sin, 2002). The ability of
single-cell electroporation to co-deliver multiple molecules, including multiple
genes to any cell type, makes single-cell electroporation a powerful and versatile
method.
With Axon Instruments’ Axoporator 800A it is possible to electroporate
individually targeted cells. Axoporator 800A provides precise control of the
voltage pulse and train parameters. The result is optimized targeted delivery of ions
or molecules into cells in culture, tissue slice or in vivo.
Introduction

Functional Checkout •5
Chapter 2
Functional Checkout
Before connecting your Axoporator 800A to an electrical outlet read
Important Safety Information on page 69. The mains power cord must be
plugged into an earthed mains outlet. Inspect the mains power cord. If it’s
damaged, then replace it.
This section describes some simple tests to quickly check that the Axoporator 800A
is operating correctly. After completing the Functional Checkout we strongly
recommend that you thoroughly read the Reference and Tutorial sections of the
manual.
Make sure that the Axoporator 800A disconnected from its power supply. Connect
the AP-1A headstage to the Headstage input connector at the rear of the Axoporator
800A. Connect the SCE-1U model cell as shown in Figure 1. The 1 mm pin of the
SCE-1U must make a firm connection to the 1 mm input socket at the front of the
AP-1A headstage. Make sure that the toggle switch on the SCE-1U model cell is
set so that 10 Mis selected and that the black ground wire provided with the
Axoporator 800A connects the 2 mm ground sockets of the model cell and of the
AP-1A headstage (rear input surrounded by yellow insulator). With the power
switch of the Axoporator 800A in the Off position, connect the power supply to the
12-14 VDC input.
Functional Checkout

6 •Functional Checkout
Switch on the Axoporator 800A, the version of the firmware will be displayed
followed by the settings display.
Look in the upper right hand corner of the panel display. It provides the value of
the feedback resistor of the AP-1A headstage connected to the Axoporator 800A. If
the AP-1A-1MU is connected, the reading will be 1M. If the AP-1A-0.1MU is
connected, the reading will be 0.1 M. In the center of the display near the top, the
resistance of the SCE-1U model cell is displayed. This reading should be 10 M.
Press and hold the Reset button (upper left hand corner of the front panel controls).
The Pulse Type (lower left hand corner of the front panel display) should show a
positive going square wave. If not, use the Pulse Type button (bottom row of front
panel controls) to select a positive going square wave. Use the Single/Train button
to select a single pulse. The lower right hand corner of the display will show a
single positive going pulse. Adjust the Pulse Voltage knob (far left row of front
panel controls) so that the display voltage reads 10.0 V and adjust the Offset
Voltage knob (beneath the Pulse Voltage knob) so that the DC offset reads 0.0 V.
Adjust the Pulse Width (far right row of panel controls) until the display reads
100 ms. Press and hold the Trigger button (bottom right of front panel controls).
The current reading (upper left of the display) should be 100 ±1 nA and the power
reading (near the center of the display) should be 1 ±0.01 µW. Terminate the test
by removing your finger from the Trigger button.
Leave all settings as above but press the Single/Train button so that two rectangular
pulses appear in the lower right hand corner of the display. Adjust the Train
Duration knob (bottom row of front panel controls) until the display reads 10 s.
Adjust the Pulse Frequency knob (next to the Train Duration knob) until the display
reads 1.0 Hz. Briefly press the Trigger button. The dual rectangular pulse in the
lower right hand corner of the display will be replaced with the word ACTIVE.
The current and power readings should be I = 100 ±1 nA and 1 ±0.01 µW,
respectively. Switch toggle on the SCE-1U model cell so that the resistance is
15 M. The current and power readings should now be I = 67.7 ±1 nA and
0.67 ±0.01 µW, respectively.
Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

Functional Checkout •7
Figure 1. Connections between the AP-1A headstage to the SCE-1U model cell.
Functional Checkout

Installation •9
Chapter 3
Installation
Standard Configuration
The standard configuration requires a microscope, micromanipulator and
Axoporator. For an example configuration you can consult the Axoporator 800
animation available at www.axoporator.com. Some sources for the components of
the basic system are listed in our Knowledge Base at:
http://www.axon.com/mr_Axon_KB_Article.cfm?ArticleID=433
Before connecting your Axoporator 800A to an electrical outlet read
Important Safety Information starting on page 69.
Micromanipulator
The micromanipulator must provide appropriate access to the biological specimen
when viewed through the microscope. For cells in dissociated cultures or slice
preparations, manipulators with fine and course adjustments are required. Coarse
manipulators are sufficient when target cells need not be directly visualized, such as
in vivo preparations and tissue slices where any cell within a region is a target.
Installation

10 •Installation
Microscope
Selecting the most appropriate type of microscope is also dictated by the
preparation and whether the target cell must be visualized. For single-cell
electroporation of cells in dissociated cultures, a high-magnification inverted or
upright microscope is needed to identify the target cell and to visualize the
micropipette tip and target cell interactions. Direct visualization of the micropipette
tip with the target cell and cell response greatly improves electroporation success.
However, the limited working distance of high magnification microscopy may
hinder rapid movement between target cells in tissue slices and in vivo when a cell
is approached blindly. In this case, a low magnification microscope is suitable;
since the micropipette tip and target cell interactions need to be directly visualized.
Instead, the micropipette tip is inserted into a region of tissue thought to contain the
cell bodies of the target cells. Successful electroporation then depends on the
probability that the micropipette tip makes contact with the target cells. While
blind targeting using low magnification objectives will reduce electroporation
efficiency, the long working distances of these objectives allow rapid movement
between sites in the preparation. Therefore, lower efficiency can be partially offset
by the high number of electroporation attempts. For in vivo preparations, dissection
microscopes offer sufficient magnification along with optimal working space for
pipette movement.
Epifluorescent microscopy is extremely useful when electroporating fluorescent
dyes to test the effectiveness of micropipette tips to be used to deliver other
molecules.
Connections
The essential connections involve the power supply and AP-1A headstage.
Power Supply
The power supply brick is connected to the 12-14 V input on the rear panel of
the Axoporator.
Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

Installation •11
Headstage
The AP-1A headstage mates with its connector on the rear panel. To complete
the circuit a ground electrode appropriate for the preparation must be used. A
Ag/AgCl pellet assembly is provided with the Axoporator. You may wish to
make your own ground electrode. The ground electrode may be a chlorided
silver wire dipped into the media bathing a cell culture or an alligator clip
placed on an incision of an in vivo preparation. The ground electrode must be
in electrotonic contact with the electroporation micropipette, but the distance
between the two is not important. Connect the ground electrode to the back of
the headstage unit. Use the 2 mm plugs or leads with 2 mm plugs to fashion a
connector to run from the ground electrode to the back of the headstage.
Optional Configurations
Additional inputs and outputs that extend the flexibility of the Axoporator are
available as well.
• To follow the voltage or current signals at the micropipette connect to the
Voutput and Ioutput, respectively.
• To use the audio monitor, plug in a headset or external speaker.
• To trigger a pulse or pulse train with an external TTL device use the
EXT. TRIGGER input.
• To trigger the Axoporator via foot connect the FOOT SWITCH.
• To apply command from an external waveform generator use
EXT COMMAND.
• To synchronize the monitoring device to the 100 Hz resistance pulses and
electroporating pulses use SYNC OUTPUT.
Oscilloscope
An oscilloscope will prove useful should an external waveform generator be used.
See Reference Section Rear Panel Inputs/Outputs for details.
Installation