axoporator 800a

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://www.moldev.com/support

http://support.axon.com/

i

PLEASE 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 Rf 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


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 MΩ 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 MΩ.

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 MΩ. The current and power readings should now be I = 67.7 ±1 nA and 0.67 ±0.01 µW, respectively.


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

http://www.axoporator.com/

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.


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

Reference Section • 13

Chapter 4

Reference Section

Audio Monitor The audio monitor is a voltage controlled oscillator that provides auditory feedback for changes in the micropipette tip resistance.

It may be used for determining when the micropipette has entered the preparation or detecting the increases in tip resistance as it comes into proximity to the cell membrane.

The output is monitored via the Headphone output on the rear panel. The audio monitor settings are user selectable (See Figure 6).

• The audio output determines the volume. (Menu 1)

• The starting pitch level sets the VCO offset. (Menu 2)

• The sensitivity setting adjusts the rate at which the audio tone pitch changes. (Menu 2)

Reference Section

14 • Reference Section

Headstage

High Voltage Precautions The voltage at the input connector of the AP-1A headstage can reach ±100 V. Never touch the input of the headstage when the Axoporator is switched on. See Important Safety Information pages 69 before connecting the headstage to the Axoporator 800A.

Static Precautions If you are in a laboratory where static is high (i.e., you hear or feel a discharge when you touch things), you should touch a grounded metal object immediately before touching the AP-1A headstage.

Hot Plugging Never connect the headstage when the Axoporator 800A is switched on.

Types Axoporator 800A has two types of headstages:

AP-1A-1MU with a feedback resistor, Rf of 1 MΩ

AP-1A-0.1MU with a feedback resistor, Rf of 0.1 MΩ

Never attempt to connect any other headstage or device to the headstage input of the Axoporator 800A.

Voltage Clamp Circuit Axoporator uses a voltage-clamp circuit illustrated in Figure 2. This voltage-clamp circuit offers great stability and bandwidth.



Figure 2. Schematic of single cell electroporation. Rf=feedback resistance, Re=electrode resistance, Rc=cleft resistance. Vin=command voltage, Vm=voltage at surface of membrane. Vo1= output of amplifier A1. Vo2=output of amplifier A2.

The operational amplifier to which the micropipette is connected is stable and forms a linear current-to-voltage converter for any combination of Re and Rc values.

The operational amplifier acts to keep its negative input equal to its positive input. Thus the voltage at the top of the pipette is equal to Vin. By the action of the simple resistive divider formed by Re and Rc, the voltage at the membrane in the vicinity of the pipette tip is Vin(Rc/(Re+Rc)). In a typical example, Vin = -6 V, Rc= 6 MΩ and Re = 30 MΩ, resulting in a voltage at the outer surface of the membrane (Vm) of about -1V. Clearly, increasing Rc will result in a higher fraction of Vin being delivered to the cell surface. This is accomplished by pressing the micropipette tip against the cell.

A second operational amplifier subtracts the command voltage from the current-to-voltage converter output to give a voltage equal to -IRf and, therefore, provides an accurate measure of the current flowing through the micropipette. At the Vin input, waveforms of various voltages, durations, and frequencies are applied.

Reference Section


Noise Current noise decreases as the value of the feedback resistor, Rf, increases. So, the current noise of the optional AP-1A-0 .1MU (Rf = 0.1 MΩ) is greater than that of the AP-1A-1MU (Rf = 1 MΩ).

Mounting For maximum mechanical stability, the AP-1A headstage should be mounted directly to the head of the micromanipulator using the acrylic mounting plate. Mounting rods are provided but this offer a less stable mounting platform.

Bath Connection The bath (or ground) electrode should be connected to the gold-plated 2 mm socket on the rear of the headstage. The bath should not come in contact with any other ground.

Electroporating while Recording Electroporating a cell while recording from it has not been attempted. It is possible that large voltage pulses would damage the recording headstage. It is also possible that the recording noise would increase during when an electroporating pulse is in progress.

Cleaning Switch off the Axoporator 800A and disconnect the headstage from the Axoporator 800A. Wipe the headstage connector with a damp cloth to clean salt spills. Avoid spilling liquids on the headstage. The Teflon input connector and gold input socket should be kept clean. Effective cleaning can be accomplished by swabbing carefully with deionized water. Occasionally, it may be necessary to add a small volume of isopropyl or ethyl alcohol to the gold input socket. Wait about ten minutes and insert a fine wick of cotton into the socket to dry the input socket.



Front Panel Controls Six push buttons and six knobs comprise the front panel controls.

H IG D E F

A

B

C

L

K

J

Figure 3. Front Panel Controls.

A: ABORT/RESET – Brief press aborts the pulse or pulse train. Press and hold aborts pulse or pulse train, clears counters and resets clock.

B: PULSE VOLTAGE – Sets the amplitude of the pulse. Electrode pulse voltage ranges from +/-100 V with 0.1 V steps throughout this range. The maximum voltage is decreased by the amount of offset voltage applied. Display dims with out-of-range values.

C: OFFSET VOLTAGE – Sets the amount of constant voltage that prevents leakage of changed molecules.

D: MANUAL COUNT – Independent counter that increments count by one each time the button is pressed. Allows one to keep track of events other than trigger activation such as the number of cells electroporated.

E: PULSE TYPE – Selects pulse type: rectangular, bipolar and bi-level. Rectangular and bi-level pulses can be selected as either positive or negative.

F: TRAIN DURATION – Sets the duration of trains when pulse trains are selected. Train durations can last between 10 ms and 100 s.

G: PULSE FREQUENCY – Sets the frequency of the pulses. H: SINGLE/TRAIN – Toggles between single pulses and trains. I : TRIGGER – Activates single pulse or pulse train. J: PULSE WIDTH – Sets the duration of the pulse. Pulse width can be varied from 200 µs to

1000 ms. When bipolar pulses are used, the minimum duration is 400 µs. K: MENU Dial – Changes values selected by the MENU button. L: MENU Button – Selects from two sets of menu options. The values for each menu item can be

adjusted by turning the menu knob. Briefly pressing the menu button steps through the first menu. Pressing and holding down the MENU button shifts to the second set of menu items.

Reference Section


Front Panel Display When an electroporating pulse or pulse train is not active the Axoporator display screen provides an update of the resistance measured at the tip of the micropipette. This resistance is automatically updated. A ±300 mV peak-to-peak voltage is delivered to the micropipette at frequency of 100 Hz. This voltage pulse and the resulting current are used by the Axoporator to calculate the resistance. As the micropipette touches the cell membrane the resistance increases and this is reflected on the Axoporator display screen.

When an electroporating pulse or pulse train is active the Axoporator display screen provides a measure of the current and the power of the pulse or pulse train. For example, if a 10 V step is applied to a 10 MΩ micropipette, then one would expect the resulting current to be 1 µA and power to be 10 µW. However, this does not account for the duty cycle. If the pulse width is 300 ms and the pulse frequency is 1 Hz, then the duty cycle is 0.3 or 30%. The Axoporator automatically accounts for the duty cycle. Thus, the current and power are displayed as 305 nA and 3.05 µW, respectively. Note that a single pulse is considered to have duration of 1s. The Axoporator will not follow a trigger that is faster than this. If you would like to calculate the peak current of a single pulse, simply divide the current reading by the duty cycle.



G

H A

D

C

B

IJ

F E

Figure 4. Front Panel Display.

A: Reports Current – When current cannot be computed question marks appear. B: Voltage – Voltage amplitude of pulse. C: Offset Voltage – Constant voltage applied. D: Manual Count – Tracks number of times manual counter pressed. E: Time – Registers total time in hours:minutes:seconds that the Axoporator has been powered on. F: Automatic Count – Tracks number of triggers. Count increments by one each time a train or pulse is triggered. G: Active – Reports “Active” when stimuli are being delivered and Single/Train selection at other times. H: Rf – Indicates the nominal value of the feedback resistor for the headstage in use. I: Total Power output of a single pulse or train is presented in µW. When power cannot be computed four arrows

appear. Blank when an external waveform generator is used. J: Micropipette Resistance (MΩ) – Provides measure of the sum of the micropipette and cleft resistances derived

from a waveform (100 Hz square wave of +/-100 mV peak-to peak) that is continuously active in the absence of triggered pulses. If resistance is < Rf four downward directed arrows appear. If resistance is > 999 Rf four upward directed arrows appear.

Reference Section


F Trains

A Pulse Types

C

E Single Pulses

B

D

Figure 5. Pulse Parameters.

A: Pulse Types – Indicates selected pulse type: rectangular, bi-level and bipolar. B: Train Duration – Indicates the train duration setting. C: Train Frequency – Indicates the frequency setting for train. D: Pulse Width – Displays the selected width for pulses. When trains are selected, the width is the duration of a

single pulse in the train. E: Pulse Type – Displays a representation of the selected pulse type. F: Train – When trains are selected this display shows twin representation of the selected pulse types.

The individual pulses within a train are defined by type, amplitude and duration in the same way that single pulses are defined. In addition, when trains are selected, the train duration and pulse frequency must be set. Pulse frequency, pulse width and train duration are all interrelated. If an out-of-range setting is made, the value selected will dim on the display.



Figure 6. MENU Options.

Menu 1 Contrast: Indicates the screen contrast setting. Volume: Indicates the intensity setting of the audio output. ‘b/a’ V-ratio: Changes the ratio relationship of the amplitude of level ‘b’ with respect to the amplitude of level a of a bi-level pulse. External Command: Disables the internal waveform generator of the Axoporator when Ext Cmd is set to On. Menu 2 VCO Offset: Indicates starting pitch level of audio tone. Sensitivity: Indicates the rate at which the pitch of the audio tone changes. Inverse: Indicates 0 when black-on-white is selected and 1 when white-on-black selected. Backlight: Indicates duration in seconds that backlight is to remain on when the instrument is switched on but not in use.

Reference Section


Rear Panel Inputs/Outputs

Figure 7. Inputs/Outputs, top row.

EXT. TRIGGER: TTL trigger input option for use with external signal generator.

SYNC OUTPUT: TTL trigger output option for triggering an external device.

Headphone: The headphone jack can be plugged into standard speakers. This audio output generates a change in frequency in response to changes in the micropipette tip resistance.

FOOT SWITCH: Trigger input for foot switch accessory.

Sync Output: Primarily designed for synchronizing the input trigger of a monitoring system such as an oscilloscope. There is trigger pulse that coincides with that of the resistance measurement frequency, 100 Hz and that of the pulse frequency. The Sync Output puts out a pulse of 100 Hz even when an external command is employed.

For automated triggering of the Axoporator the Ext. Trigger input can be used. This can be used in place of the Trigger button or Foot Switch of the Axoporator. The upper limit of a 50% duty cycle still applies.



Figure 8. Inputs/Outputs, bottom row.

VOUTPUT: Electrode voltage (Vo) output. Provides a 0.1V signal for 1V delivered to micropipette.

EXT COMMAND: External command option for external waveform generator.

Although the three pulse types provided with the Axoporator have proven to be more than sufficient for single-cell electroporation, a signal generator can be used to deliver virtually any waveform command to the headstage. The signal amplitude of the waveform must not exceed ±10 V. When EXT COMMAND is enabled this will be indicated on the Axoporator display screen.

1. Selecting the external command mode only disables the internal waveform generator of the Axoporator.

2. An external command always sums with the internal command, even if the external command mode is set to Off. Thus, no external signal generator should be connected to the Axoporator, unless it is to be used as the only source of a command.

The resistance, current and power readings on the display screen will not be valid when the external command is active. Successful single-cell electroporation is critically dependent on the micropipette tip voltage and current. So, VOUTPUT and IOUTPUT should be used to monitor the command and the current response of the micropipette. A poor quality current response, drooping waveform, may lead to sub-optimal electroporation. Also, the resistance at the micropipette tip must be monitored to ensure that the tip and cleft resistance remain optimal. The resistance is computed using Ohm’s Law.

IOUTPUT: Current output scaling dependent on headstage: 1V/1 µA or 0.1V/1 µA.

Reference Section


HEADSTAGE: Connector for AP-1A headstage.

For Factory Test Only: For factory test procedures.

12-14 VDC: Connector for external power supply.

On/Off switch



Overview of Pulses and Trains The Axoporator 800A provides three basic pulse types: rectangular, bipolar and bi-level. Pulse amplitude and timing are set via the intuitive control interface discussed in the Front Panels Controls portion of the Reference Section.

Train duration

Pulse width

1/frequency

b

a

Positive rectangular pulse

Bipolar pulse

Positive Bi-level pulse

Sync

Figure 9. Axoporator pulse types.

Reference Section


Figure 9 shows the three basic pulse types available from the Axoporator, basic pulse timing terminology and the relationship between pulse activation and the sync pulse. The pulse width of a standard positive-going rectangular pulse and bipolar pulse are compared. During the bipolar pulse the pulse amplitude is positive for half the pulse width and negative for other half. The dotted vertical line shows the timing relationship between the pulses and the synchronized TTL output. This output can be used to synchronize the display of an optional pulse monitoring system.

Simple rectangular pulses are effective. Their duration can range from 200 µs to 1000 ms.

Bipolar pulses have two components of equal amplitude and opposite polarity. Bipolar pulse duration can range from 400 µs to 1000 ms.

Bi-level pulses are composed of two components, ‘a’ and ‘b’. Component ‘a’ is of large amplitude and short duration, while component ‘b’ is of smaller amplitude and long duration. The order of presentation within the pulse is component ‘a’ followed by component ‘b’, or vice versa. The polarity of the two components is the same and either negative or positive polarity can be selected. The voltage amplitude of component ‘a’ is selected first. The amplitude of component ‘b’ is determined by changing the ‘b/a’ V-ratio. The total bi-level pulse duration is selectable: the duration of component ‘a’ is 2% of the total pulse, and the duration of ‘b’ is 98%. Thus, the duration of the bi-level pulse is the sum of the two components. For instance, if a 100 ms pulse width is selected, then duration of component ‘a’ is 2 ms and component ‘b’ is 98 ms. The total pulse duration of bi-level pulses can range from 10 ms to 50 s.

After selecting the pulse type and the pulse polarity, one must decide whether to deliver a single pulse or a train of pulses. Train frequencies range from 1 Hz to 2 kHz. “Optimal” parameters should rapidly transfer molecules into the cell with minimal perturbation.

The entire procedure, including micropipette placement and electroporation should not take more than a few tens of seconds.



Terminology Frequency equals the inverse of the duration between the start of two successive pulses in the train.

Period is the time between beginning of one pulse and the start of the next.

Period = 1/Pulse frequency

Duty cycle = Pulse width/Period

Number of pulses in train = Train duration/Period

Total on-time of pulses in train = Duty cycle ∗ Train duration or Pulse width x number of pulses in train.

Using Axoporator’s Pulse Generator The maximum duty cycle is 50%. Stated differently, the period cannot be less than double the pulse width. (With an external pulse generator you can increase the duty cycle.)

Train duration must be at least equal to the pulse width.

If the train duration is to remain fixed, then the pulse duration must decrease as pulse frequency increases.

Sample problems:

1. Calculate the total on-time, number of pulses in the train, the duty cycle and period given the following parameters:

10 ms pulse width

3 second train

10 Hz pulse frequency

Period = 1/pulse frequency = 1/10 Hz = 100 ms

Reference Section


Duty cycle = pulse width/train period = 10ms/100ms = 0.1

Total on-time = duty cycle ∗ train length = 0.1 ∗ 3s = 300 ms

Number of pulses = train duration/period = 3000 ms/100 ms = 30

2. If a 300 ms on-time is desired with a 1 ms second pulse width, then how should the pulse frequency be set to achieve a train duration of 3 seconds? How many pulses will there be in the train?

First calculate the duty cycle = total on-time/train duration = 300 ms/3000 ms = 0.1

Next calculate the period = pulse width/duty cycle = 1 ms/0.1 = 10 ms

Finally calculate the pulse frequency = 1/period = 1/10 ms = 100 Hz

Number of pulses in the train = train duration/period = 3000 ms/10 ms = 300

Triggering Pulses are triggered by pressing the front panel TRIGGER button, by pressing the footswitch, or by an external input.

Micropipette Holder The HL-U series holder provides a universal fit for a very wide range of glass pipette diameters and will fit any of the U-type headstages from Axon.

Holder Design The barrel of the holder is made of polycarbonate. There are two different barrel lengths (16 mm and 28 mm). The shorter length may provide the needed clearance between the headstage case and other components in the experimental setup.

Mechanical stability of the micropipette is assured in several ways. (See Figure 10.) As the pipette cap is closed, the cone washer is compressed on the micropipette from the force applied to the front and back of the cone washer. The



cap also forces the blunt end of the microelectrode against the rear wall of the holder bore. (The micropipette should always be inserted as far as it will go in the holder.) The holder mates with the threaded Teflon connector on U-type Axon

Figure 10. Exploded view of the HL-U holder.

headstages and is secured in place with a threaded collar.

The bore size of the HL-U ameter (OD) of

ear), 1.5 mm (orange) and 1.7 mm (clear). When the

rs no greater stability than properly chlorided silver wire.

: one 50 mm length of silver wire,

s.

accepts pipettes with an outer di1.0-1.7 mm. Micropipettes are secured by a cone washer with an inner diameter (ID) that accommodates the pipette OD. Color-coding aids identification of the four sizes of cone washers:

1.0 mm (orange), 1.3 mm (clpipette OD falls between two sizes of cone washers, the larger size cone washer should be used. For instance, if the pipette OD is 1.6 mm, then use a cone washer with an ID of 1.7 mm.

An Ag/AgCl pellet offeMoreover, the diameter of the pellet (1 mm) restricts its use to pipettes with a large ID (> 1.1 mm). Therefore, the HL-U is supplied with 0.25 mm silver wire, which must be chlorided before use. (See Figure 11.)

Spare components included with each holder are40 cone washers (10 of each size), and one 70 mm length of silicone tubing. Cut into 2 mm lengths, the silicone tubing will yield approximately 30 replacement silicone seals. Additional cone washers, silicone tubing, pins and silver wire can be purchased from Axon Instruments, as well as optional Ag/AgCl pellet assemblie

Reference Section


Optional Ag/AgCl Pellets

The HL-U holder will accommodate a 1 mm diameter Ag/AgCl pellet that should remain stable for many months. The inner diameter (ID) of the pipette must be > 1 mm. A wax-sealed Teflon tube surrounds the silver wire. This ensures that the electrode solution only contacts the Ag/AgCl pellet. Three pellet assemblies are sold as HLA-003.

Figure 11. Ag/AgCl pellet assembly.

Holder Use

Insertion of Electrode

e the pipette cap is loosened so that pressure on the cone washer is ove the cap. Push the back end of the micropipette

ds of the

the pipette should be filled with solution. The chlorided tip of the wire should be inserted into this solution. Avoid wetting the holder since this will increase the noise.

Make surrelieved, but do not remthrough the cap and cone washer until it presses against the end of the bore. Gently tighten the cap so that the micropipette is gripped firmly.

To minimize cutting of the cone washer by the sharp back end of the micropipette, you can slightly smooth the edges by rotating the enpipettes in a Bunsen burner flame prior to pulling.

Filling Electrodes

Only the taper and a few millimeters of the shaft of



Silver Chloriding

It is up to you to chloride the end of this wire as required. Typically the chlorided wire will need to be replaced or rechlorided every few weeks. simple yet effective chloriding procedure is to clean the silver wire do

A wn to the

paper and immerse the cleaned wire in bleach for until the wire is uniformly blackened. This provides a

ets d

Holder Maintenance

required, briefly wash in ethanol or mild soapy water. Never use methanol or

the Silver Wire

of the barrel making sure that the silicone seal is flush with collar over the back of the barrel. With

ected toward the bent-over wire, screw the pin cap .

AdaHLRhea

bare metal using fine sand about 20 minutes,sufficient coat of AgCl to work reliably for several weeks. Unstable offsduring experiments are suggestive of the need for rechloriding. The chlorideregion should be long enough so that the micropipette solution does not come in contact with the bare silver wire.

Heat smoothing the back end of the electrode extends the life of the chloride coating by minimizing the amount of scratch damage. Another way to protect the AgCl coating is to slip a perforated Teflon tube over the chlorided region.

Cleaning

Frequently rinse the holder with distilled water. If more thorough cleaning is

strong solvents.

Replacing

To replace the silver wire, insert the nonchlorided end through the hole of the silicone seal and bend the last 1 mm of wire over to an angle of 90°. Press the wire into the backthe back of the barrel. Slip the threadedthe large end of the pin dirdown firmly, but without excessive force. This assures good electrical contact

pters -U right-angle adapters allow the HL-U series holder to emerge at 90° from the

dstage. Use the HLR-U with the HL-U holder.

Reference Section


HLB-U BNC-to-Axon adapter allows push-in style BNCs to be connected to the

ard accessory provided with the Axoporator 800A. It is useful for the Functional Checkout and testing.

as a 1 mm pin that connects to the AP-1A input. An unlabeled the

rear Two resistance settings, 10 MΩ and 15 MΩ, are available.

input of the AP-1A headstage.

Model Cell • SCE-1U model cell is a stand

The model cell h2 mm gold socket on the model cell connects to the 2 mm grounding socket on

of the AP-1A headstage. The circuit is shown in Figure 12.

Figure 12. SCE-1U circuit.


Tutorial • 33

Chapter 5

Tutorial

Guidelines for Single-Cell Electroporation Successful single-cell electroporation is highly dependent on three factors: the geometry of the glass micropipette tip, proximity between the micropipette tip and the target cell, and the stimulation parameters.

Micropipettes

Fabricating Micropipettes

In general, tip diameter should be much smaller than the cell diameter, in the range of 0.5 µm. The geometry of the micropipette tip shank may also be an important consideration.

Micropipettes of the type described here will work for almost any cell type tried to date. Use capillary glass whose internal diameter is 1.15 to 1.2 mm and outside diameter is 1.6 to 1.7 mm with a glass filament fused to its interior to facilitate tip filling. Inexpensive glass like Corning Pyrex or Kimble Kimax is available from a variety of major suppliers. Purchase 4" pieces to pull two similar length micropipettes of adequate length. It is recommended that the back ends be flame-polished to prevent the sharp end from cutting the washer within the micropipette holder.

Tutorial

34 • Tutorial

Use a single-stage pull so that the micropipette tips look more like those an intracellular electrode than a patch electrode. This shape has an added benefit in tissue slices or in vivo preparations by limiting the damage to the surrounding tissue. Micropipettes should have tips about 0.5 µm in diameter and should have a resistance of 35 to 40 MΩ when filled with a physiological salt solution or its equivalent. There is currently no evidence that fire polishing the tip contributes to successful single-cell electroporation. If you have any reservations about fabricating your own micropipettes or do not have access to pipette puller, then you may wish to consider a commercial source of prefabricated, custom micropipettes, such as Precision Microdevices ([email protected]).

Filling the Micropipette

Only a few microliters are needed to fill the micropipette tip. As a consequence, a very small volume of the solution containing the molecules to be electroporated is needed. Filling is typically done at the back of the micropipette either by immersing it in a small volume of the solution or by injecting the solution from a syringe. A less desirable filling method would be to immerse the microelectrode tip into the solution containing the molecules to be electroporated and apply suction to the back of the micropipette to fill the tip. This would be an extremely slow process for viscous solutions.

Typically, the molecules to be electroporated are dissolved in physiological salt solution and this same solution is used to backfill the micropipette. The solution used to fill the micropipette may either closely mimic an intracellular or extracellular solution. Extracellular solution simplifies the composition of the solution in the cleft but intracellular solutions might seem better for ensuring cell health. To date there is no evidence that either is preferred.

DNA solutions are usually made up in sterile water and are not sufficiently conductive for electroporation. Therefore the stock DNA solution should be combined with a physiological salt solution. When working with precious molecules it is important to consider the preparation of solutions as well as the


mailto:[email protected]

Tutorial • 35

amount of solution loaded into the micropipette. Compare the two methods outlined below.

1) 1 µg of plasmid DNA is added to 5 ml of a standard physiological salt solution; a DNA concentration the same as for most standard lipofection techniques. 2 µl is used to load the micropipette.

2) 1 µl of plasmid DNA (1 µg/µl) is added to 29 µl of standard physiological salt solution.

Both of these techniques use DNA sparingly. In the former, the total DNA in the pipette is about 0.4 ng whereas in the latter it is 67 ng.

Of course, the filling solution need not contain genes. This technique should work for electroporating small peptides, drugs, dyes, calcium ions and probably even a variety of uncharged molecules. Filling is generally done in two stages.

1. Use a hand-held pipetter with a 10 µl disposable tip. Add 2 to 3 µl of the solution to be electroporated to the back of the micropipette. The solution moves along the internal glass filament by capillary forces and fills the tip. It may take a few taps of the glass to get the flow started. After 30 seconds or so, apply a few gentle taps near the shank to dislodge bubbles.

2. Use a 1 ml syringe to backfill the micropipette with the solution used to dissolve the molecules to be electroporated. You should start adding the solution several millimeters behind the tip and fill to about the middle of the pipette length. Gently tapping the micropipette shank will dislodge the air bubble behind the tip.

Needle-tubing combinations that work with standard syringes are available from commercial suppliers such as:

http://www.smallparts.com/components/

http://www.wpiinc.com/WPI_Web/Lab/MicroFil.html

Tutorial

36 • Tutorial

To make your own syringe with thin tubing, remove the internal plunger. Place your hands on opposite ends of the syringe and rotate it while heating its midsection over a small flame. Once the plastic begins to melt, remove the syringe from the flame and pull the ends apart. Gentle pulling will produce a long, thin plastic tube. After cooling, cut this tube such that a 7 to 10 cm section extends from the back end of the syringe. Reinsert the plunger and make sure that the tube will allow solution to flow through it.

Mounting the Micropipette

When the micropipette is inserted into the holder, the Ag/AgCl wire need only be immersed into the solution far enough for electrical contact to be made. There is no reason to have the wire close to the tip of the micropipette. Loosen the pipette cap of the holder. Feed the silver wire into the filled micropipette and push the micropipette through the pipette cap into the barrel of the holder. Carefully tighten the pipette cap so that the micropipette is held in place. Attach the HL-U holder to the AP-1A headstage that is mounted on a micromanipulator.

Positioning the Micropipette Tip

While the current is measured, the micropipette tip is slowly advanced against the cell membrane (identical to a standard patch-clamp measurement at the time of seal formation). As the pipette pushes against the cell and increases the cleft resistance, the current falls as the sum of the pipette and cleft resistance increases. A voltage divider forms by the resistances of micropipette tip and the cleft between it and the cell. When resistance has gone up 33%, then the current drops by 25%. This results in a voltage (Vm) at the cell surface that is about 25% of the commanded voltage. Presumably it is the voltage at this location that is responsible for creating the lipidic channels across the cell membrane through which genes, peptides, dyes, and most other compounds can flow. Although higher degrees of indentation result in a larger fraction of the pipette voltage being applied at the cell surface, there is presumably a trade off between the increased effectiveness and damage to the cell. It remains to be


Tutorial • 37

demonstrated whether increasing the resistance upon indentation to greater than 33% will increase the effectiveness of single-cell electroporation.

Figure 13. Micropipette tips of three different sizes are shown pushing against a cell membrane when they (upper right) first contact the cell and (upper left) after they are advanced an additional micron. Magnified views of the regions in the rectangles are shown in the lower half. The electrode is at a 45-degree angle, common for a patch clamp setup. The thickness of the cell is drawn to scale.

It is important to note that the site of electroporation need not be restricted to the cell soma, and can be directed at any portion of the cell. This can be useful for those studies using polarized cells, such as photoreceptors or those cases where it is desirable to induce an initial localized increase of the molecules electroporated.

Not surprisingly, the efficiency of single-cell electroporation declines when it is not possible to visually direct the micropipette tip to the cell membrane. However, under these less than optimal conditions, single-cell electroporation

Tutorial

38 • Tutorial

can still be successful. At least this appears to be so for central nervous system tissue, if higher voltages are used.

Selecting the Stimulation Parameters The polarity of the voltage pulse should match the polarity of the charge on the molecules to be delivered to ensure that there is an electrical driving force to move the molecules towards the cell. To keep the molecules from leaking from the micropipette the offset or holding potential should be opposite to that of the molecules to be electroporated. Even though the appropriate polarity can be predicted, it should be tested empirically. For example, ‘anionic’ fluorescent dextrans often move as cations following repeated single-cell electroporation stimuli. For dyes, the best way to optimize the pulse protocol is by watching their movement.

Voltage pulse widths from a few µs to tens of ms form pores. These pores form in a few microseconds and close more slowly. Thus, the pulse protocols to produce electroporation are quite simple. For most applications, simple rectangular pulses suffice. The pulses must result in total channel open time sufficient for an effective concentration of molecules to enter the cell. Not surprisingly, several different combinations of pulse widths, frequencies, and train durations may work equally well. For further flexibility an external waveform generator can be used.

Pulse amplitude and duration influence both the size of pore formation and the distance that molecules of different motility can move. The size of the transport molecules is particularly relevant when selecting a pulse protocol since electrophoretic mobility through biological tissue is highly size-dependent. Smaller molecules like fluorescent dextrans are electroporated using single, low-voltage pulses, while larger molecules like DNA require multiple pulses at higher voltage. Even so, fluorescent dyes are useful for testing micropipettes to be used for electroporation of DNA, since tips with a geometry successful for dye transfer also work well for DNA. In fact, 250 kD dextrans require electroporation conditions similar to those for DNA.


Tutorial • 39

Electroporation of DNA that leads to gene expression requires a multi-step process. After moving through the pores in the cell membrane, DNA find its way across the nuclear membrane by a mechanism that is not fully understood. In general, electroporation of DNA requires voltage pulses of greater amplitude and longer duration than for smaller molecules. Trains of high voltage, short duration rectangular pulses work well for DNA electroporative transfection in several cell types. Bi-level pulses have also been shown to work as well.

Depending on the micropipette tip resistance and pulse characteristics, the micropipette may fuse with the cell membrane. Fusion between the tip and the cell has been characterized in detail for cultured α-TN4 cells (a lens epithelial cell line). It occurs when the average power dissipated by current flow through the tip and cleft resistances exceeds 0.6 µW. Few cells survive the fusion. The attachment is sufficiently strong that a tissue-cultured cell can be torn from the bottom of the culture dish as the micropipette is withdrawn. Therefore, for α-TN4 cells an additional constraint can be placed on micropipettes—average dissipated power should be 0.6 µW or less. Therefore, extreme caution is required when using low resistance (e.g., 1 MΩ to 7 MΩ) micropipettes. The 0.6 µW limit for cell damage assumes a 3 second train of 1 ms pulses at a frequency of 100 Hz. Shorter pulses at higher frequencies that lead to a reduced duty cycle, increase this power limit. Also, using longer train durations with 1 ms pulses at lower frequencies increase this limit.

Although voltage and wattage limits might not be absolute or generally applicable to all preparations these limits do provide some boundary conditions. Figure 14 shows the relationship between average power dissipation (I2R x duty cycle) and pulse voltage for a range of cleft resistances. The light gray region shows the voltage-cleft resistances that do not exceed 0.6 µW ‘limit’. The graph indicates that higher resistance electrodes are recommended.

Tutorial

40 • Tutorial

Figure 14. A family of curves showing the average power (I2R x duty cycle) dissipated by current flow through the tip and cleft resistance for a range of pipette voltages at a 0.1 duty cycle. Each curve is for a different sum of electrode and cleft resistances (in MΩ). The speckled inset depicts the range of parameters successful in producing electroporation of genes 5.6 kb or larger. The 4 V limit assumes cell indentation sufficient to increment the resistance by 33%.

As a general rule, to maximize cell loading and optimize cell health use high concentrations of the molecules to be delivered and minimize the voltage amplitude and stimulus duration.

Overview of Protocols for Loading Dissociated Cells in Culture Since the details for single-cell electroporation of various molecules are documented in the scientific literature, we present only an abbreviated list of suggestions. In each case it is assumed cell indentation by the micropipette results in a total resistance increment of 25% to 30% and that the starting micropipette resistance is 35 MΩ to 40 MΩ.


Tutorial • 41

Rectangular Pulses

Voltages of 2 V to -4 V are sufficient for low molecular weight substances such as Lucifer yellow, Alexa dyes, siRNA's, etc. The pulse polarity will have to be adjusted to be the same as that of the molecules to be delivered. The two protocols outlined below have been shown to work.

Protocol 1:

Train of 100 Hz for 3 seconds Pulse amplitude: 2 V to -4 V Pulse duration: 1 ms

Protocol 2:

Single pulse Pulse amplitude: -3 V to -4 V Pulse duration: 300 ms

For small to medium size genes (5 kb to 14 kb size range), a rectangular pulse should be tried initially. Voltages of -5 V to -10 V are useful for inserting large DNA molecules. For genes ranging from 4 to 5 kb -5.5 V to -6 V is sufficient. Larger voltages are required for larger genes. To date, 14 kb is the largest gene that has been successfully electroporated. In general, expression of the larger genes is problematic. As always, the polarity of the voltage pulse must be the same as the net charge on the gene.

Start with a pulse voltage of -6 V and width of 1 ms. Set the pulse frequency to 100 Hz and the train duration to three seconds. Use a duty cycle of 10% and a total on-time of 300 ms. If these parameters are ineffective, but the cells still seem healthy after the train is complete, consider increasing the voltage stepwise to -10 V. Cell viability may deteriorate as the voltage approaches -10 V. Instead of increasing the pulse amplitude try changing two other parameters:

Tutorial

42 • Tutorial

1) Increase the train length. Train duration of 6 to 10 seconds are quite manageable. Longer durations will make the electroporation seem tedious.

2) Double the pulse width using the same frequency and train length. Now the duty cycle is 20%.

For cells that do not tolerate the settings suggested above reduce the pulse width and increase the pulse frequency. Try a -6 to 8 V pulse with a width of 200 µs and a frequency of 2 kHz. The duty cycle will be 40%. Set the train duration from 3 to 10 seconds to achieve a total on-time of 1.2 to 4 seconds.

Bi-level Pulses

If a simple rectangular pulse protocol proves damaging to your cells, then a bi-level pulse protocol might be a suitable alternative. The bi-level pulse protocol that has worked for the electroporation of small molecules (e.g., dyes, oligos, salts) is one in which level ‘a’ precedes level ‘b’. The ‘a’ component of the bi-level pulse opens the channels and ‘b’ component drives the dye molecules into the open channels of the cell. The initial settings for this type of bi-level pulse are:

‘a’ = -20 V

b/a = 5%, that is, the amplitude of ‘b’ is -1 V

Pulse duration = 100 ms

The result is a -20 V pulse, 2 ms in duration is followed by a -1 V pulse, 98 ms in duration. A single bi-level pulse will produce visible fluorescence in the cell and a train of 3 to 5 pulses will inject enough dye for a cell to fluoresce brightly.

When a simple train of rectangular pulses fails to electroporate larger molecules, such as DNA (as plasmids), then a bi-level pulse protocol in which the ‘b’ level precedes the ‘a’ level may work. Although this protocol is useful,


Tutorial • 43

the insertion of genes larger than 6 to 8 kb remains a challenge. The actual mechanism of entry is not really known. It is tempting to suggest that the ‘b’ component causes voltage-dependent binding of the molecules to the cell membrane and then pores form in response to the large amplitude ‘a’ component. The initial settings for this type bi-level pulse are:

‘a’ pulse should be -20 but not more than -30 V ∗

b/a = adjust so that ‘b’ is between -5 to -10 V

Pulse duration = 100 ms

The result is a smaller amplitude, 98 ms pulse followed by a larger amplitude, 2 ms pulse. A train of 3 to 5 bi-level pulses is appropriate. One pulse per second is a good starting point but the time between pulses must be determined empirically.

Testing the Setup Test the setup by delivering fluorescent dye from a micropipette into saline. Fill the micropipette with a 100 µm solution of fluorescent dye, like rhodamine-dextran 3000. Place the saline solution in a small chamber, such as a 35 mm culture dish, on the stage of an epifluorescence microscope, with the ground electrode immersed in the saline. Position the micropipette in the saline and focus on the tip. Look for dye leaking from the tip under epifluorescent illumination. Molecules may leak out of the micropipette when it is in bath solution (or a tissue). There is little that can be done to counter the movement of uncharged molecules from the micropipette, other than to restrict the time that the micropipette is immersed in the larger volume. However, for those molecules that carry a charge, the Offset Voltage can be used to oppose their movement out of the micropipette. Apply a holding voltage using the Offset Voltage dial until the leak current stops. Next try a range of pulse parameters, including single pulses and trains of different pulse shapes and polarity,

∗ Pulse levels of more than -30 V can be damaging to cells even if high resistance micropipettes are used.

Tutorial

44 • Tutorial

while watching the dye. Determine which pulse parameters provide best control over dye movement. Optimal parameters should be able to deliver concise boluses of dye with no leakage.


Troubleshooting Guide • 45

Chapter 6

Troubleshooting Guide

General Rule Work through the Functional Checkout.

Symptom: High or infinite tip resistance Possible cause: Incomplete circuit.

Suggestions: Ensure that ground electrode is in contact with bath or preparation.

Test all wires and connections for disconnections or breaks.

Possible cause: Clogged micropipette.

Suggestions: Place micropipette tip in bath and apply pulses of inverted polarity.

Replace micropipette tip.

Spin down all pipette solutions to remove undissolved particles.

Symptom: Decrease in tip resistance Possible cause: Broken tip.


46 • Troubleshooting Guide

Suggestion: Change micropipette. If the micropipette tip breaks often, change pipette puller settings to form blunter tips that are stronger.

Symptom: Positional drift Possible cause: Loose manipulator or holder.

Suggestion: Tighten all holders/brackets.

Possible cause: Loose pipette.

Suggestion: Tighten or replace cone washer in pipette.

Possible cause: Manipulator drive slip.

Suggestion: Tighten manipulator components, service manipulator, or replace it.

Symptom: Contents leaking from micropipette tip Possible cause: Offset potential not set optimally.

Suggestions: Chloride silver wire in micropipette holder and use chloride internal solutions.

Adjust offset voltage.

Symptom: Unable to load cells Possible cause: Incorrect tip geometry.

Suggestions: Try smaller tip diameter.

Try fluorescent-dextran dye to find appropriate micropipette tip shape.

Possible cause: Unhealthy cells.

Suggestion: Try another source of cells or improve health of preparation.


Troubleshooting Guide • 47

Possible cause: Cell “gunk” enters tip causing a diffusion barrier.

Suggestion: Replace micropipette.

Possible cause: Inappropriate pulse protocol.

Suggestion: Review Tutorial in this chapter.

Symptom: Current or power reading is out of range Possible causes: Can’t compute correct current: I = ???A.

Can’t compute correct power reading: µW displays four arrows.

Suggestions: Decrease amplitude, frequency and or width of pulse.

Symptom: Grey out of pulse frequency or width reading Possible cause: Duty cycle requested is > 50%.

Suggestion: Reduce pulse frequency or width.

Symptom: No Rf value is reported Possible cause: Headstage is not properly connected.

Suggestion: Switch off the Axoporator 800A and check the connection to the headstage input.


Specifications • 49

Chapter 7

Specifications

Unless otherwise specified, TA = 20 ºC, 1 hr warm-up time, generated voltages are the greater of ±100 V or ±5%, generated frequency and duration are ±2% for bi-level pulses and ±1% for all other pulses.

AP-1A Headstage (voltage clamp) Main unit connection via DB-15 connector

(Connect to or disconnect from the main unit only when the power is off.)

Size: 2.25" x 1.14" x .0.87" (57.2 mm x 29.0 mm x 22.1 mm)

Mounting rod length: 102 mm (4")

Mounting rod diameters: 1/4", 5/16" (standard) or 3/8" (6.3 mm, 7.0 mm or 9.5 mm)

Specify non-standard mounting rod diameter with order.

Mounting plate: Acrylic

Ground socket: 0.08" (2 mm) diameter.

Specifications

50 • Specifications

Input socket for micropipette holder: 0.04" (1 mm) diameter.

Cable length: 3 m (10 ft)

Voltage: ±100 V (max.) at micropipette

Slew rate: 10V/µs

AP-1A-1MU (standard) Gain: Rf = 1 MΩ

Maximum deliverable to micropipette with measurement subsystem system functional:

Current: ±10.0 µA Power: 100 µW

Maximum deliverable to micropipette, correct pulse, measuring subsystem overloaded:

Current: ±30.0 µA

AP-1A-0.1MU (optional) Gain: Rf = 100 kΩ

Maximum deliverable to micropipette with measurement subsystem system functional:

Current: ±100.0 µA Power: 100 µW

Maximum deliverable to micropipette, correct pulse, measuring subsystem overloaded:

Current: ±300.0 µA



Micropipette holder HL-U holders mate to threaded Teflon input connectors of the AP-1A headstages. Post for suction tubing is 1 mm O.D. on both types of holders. HL-U holder accepts glass 1.0 to 1.7 mm OD. Supplied with silver wire. Optional HLR-U right-angle adapter and HLB-U BNC adapter are available.

Main Unit Case dimensions: 4.1" high x 10.3" wide x 7.3" deep (105 mm x 262 mm x

185 mm)

Net weight: 3.2 lbs (1.45 kg)

Operating modes

Active mode

A train or single pulse is generated in response to a trigger.

Approach mode

In this mode the system generates a low-level approach waveform for resistance measurement. Approach mode is activated when in Active mode is in operation.

Type: Square wave

Frequency: 100 Hz

Amplitude: 0.1 to 1.0 V in steps 0.1 V, above and below the Offset Voltage Fixed for each headstage type.

Manual External Command Mode

Active and Approach modes are disabled.

Specifications


Offset Voltage 0 to ±10.0 V in 0.1 V steps, always on in active, approach and manual external command modes.

Pulse types Square and bi-level pulses with positive and negative polarity, as well as bipolar available.

Pulse Generation (active mode, internal) Pulse voltage, Pulse width, Train duration and Train frequency increment in steps of 1, 2, 5 following a 10% pattern as follows:

10, 11, 12, …, 18, 19, 20, 22, 24, …, 46, 48, 50, 55, 60, 65, …, 90, 95, 100.

Pulse Voltage

±(1 to 100) V in steps following the 10% pattern. Sum of DC offset and pulse voltage does not exceed ±100V.

Bi-level pulse: Component ‘b’ to component ‘a’ voltage ratio is 5% to 95% in 5% steps.

Pulse Width

Mono-polar pulse: 200 µs to 1 s.

Bi-polar pulse: 400 µs to 1 s.

Bi-level pulse: 10 ms to 20 s. Component ‘a’ width to whole pulse width (‘a’+ ‘b’) ratio is fixed at 2% (1:50).

Train Duration

10 ms to 100 s in steps following the 10% pattern. Train always includes at least one whole pulse.



Train Frequency

Mono-polar pulse: 1 Hz to 2 kHz. Bi-polar pulse: 1 Hz to 2 kHz. Bi-level pulse: 0.024 Hz to 50 Hz. Train duty cycle: train frequency (Hz) x pulse width (s) does not exceed

50%.

LCD display For settings and readouts, 240 x 128 pixels, full-graphic with backlight, adjustable contrast.

Settings

Pulse and train parameters, DC offset, and menu options (bi-level pulse ratio, screen appearance, and audio monitor).

Readouts

All modes

Items cleared on power up or clear button:

Trigger count (0..9999) Manual count (0..9999) Elapsed time (hh:mm:ss)

Active mode

Micropipette tip resistance (MΩ) measurement

AP-1A headstage feedback resistance, Rf

Items held for 2 seconds following a return to approach mode:

Delivered power (normalized to 1 s) Delivered current (Delivered power / micropipette voltage, normalized to 1 s)

Specifications


Manual External Command Mode

When active the following readings are disabled:

Micropipette tip resistance, current and power.

Rear panel All connectors grounding wired to internal ground, and to DC power supply negative terminal. DC power supply output isolated from mains ground.

Factory Setup (DB-9F connector)

Serial connection to PC for firmware upgrades and factory testing only. Must be left disconnected during normal operation.

AP-1A Headstage (DB-15F connector)

Used only for AP-1A-1MU and AP-0.1A-0.1MU headstages.

(Connect to or disconnect from the main unit only when the power is off.)

Outputs

Ioutput (micropipette current)

Sensitivity = 1 V/µA for AP-1A-1MU headstage Sensitivity = 0.1 V/µA for AP-1A-0.1MU headstage

Output impedance = 500Ω

Voutput (voltage applied to the micropipette)

Sensitivity = 0.1 V/V Output impedance = 475Ω

Sync Output

Zero to 5 V signal, with the same width as the generated pulse (including the approach waveform), output impedance 10 kΩ. During bi-level pulse



generation contains a 5 µs notch at the boundary between components ‘a’ and ‘b’.

Audio monitor (voltage controlled oscillator)

An audible frequency output proportional to the measured micropipette resistance.

Maximum output voltage = 5 V p-p, shaped square wave

Output impedance = 1 kΩ

Attenuation = 0 to 63 db (The lower of 40 db, or last setting is power up default.)

Offset frequency at 10 x Rf = 50, 100, 200, 400, 800 Hz (200 Hz is factory default).

Inputs

Ext. Trigger (external trigger)

Positive going pulse starts internal pulse generation by switching to active mode.

Minimum width = 100 ms

Above 2.4 V accepted as logic HIGH, below 0.5 V is accepted as logic LOW.

Protected to ±15 V

Foot Switch (external trigger)

Normally open, voltage free, contact input.

Wetting current = 5 V / 12 kΩ (0.5 mA approx.)

Minimum duration = 100 ms

Specifications


Ext. Command (external voltage command)

Note: Command sums linearly with DC offset voltage.

Sensitivity = 10 V/V, input of ±10 V produces ±100 V

Input impedance = 11 kΩ

Protected to ±15 V

Power requirements (using the provided power supply) Line voltage: 100 - 264 VAC, max 0.9A Line frequency: 50 - 60 Hz DC out to main unit: 12 - 14 VDC

Model Cell SCE-1U (10 MΩ/15 MΩ switchable)

Accessories Provided Theory and Operation Manual AP-1A-1MU headstage AP-1A-0.1MU headstage (optional, may be substituted on request with order) SCE-1U (10 MΩ/15 MΩ switchable) One HL-U electrode holder Two 2 mm plugs for use with headstages Two ground wires One mounting rod Foot Switch


References • 57

References

Single-cell electroporation Haas, K., Sin, W.-C., Javaherian, A., Li, Z. Cline, H.T. Single-cell electroporation for in vivo neuronal gene expression. Neuron, 29:583-591, 2001.

Haas, K., Jensen, K., Sin, W.-C., Foa, L., Cline, H.T. Targeted electroporation in Xenopus tadpoles in vivo - from single cells to the entire brain. Differentiation, 70:148-154, 2002.

Lundqvist, J.A., Sahlin, F., Aberg, M.A., Stromberg, A., Eriksson, P.S., and Orwar, O. Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes. Proc Natl Acad Sci USA 95, 10356-10360, 1998.

Nolkrantz, K., Farre, C., Brederlau, A., Karlsson, R.I., Brennan, C., Eriksson, P.S., Weber, S. G., Sandberg, M., and Orwar, O. Electroporation of single cells and tissues with an electrolyte-filled capillary. Anal Chem 73, 4469-4477, 2001.

Rae, J.L., and Levis, R.A. Single-cell electroporation. Pflugers Arch 443, 664-670, 2002.

Sin, W.-C., Haas, K., Ruthazer, E.S., Cline, H.T. Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature, 419:475-80, 2002.

References

58 • References

General Electroporation Atkins, R.L., Wang, D., and Burke, R.D. Localized electroporation: A Method for Targeting Expression of Genes in Avian Embryos. Biotechniques 20, 93-95, 2000.

Chang, D.C. Cell poration and cell fusion using an oscillating electric field. Biophys J 56, 641-652, 1989.

Ho, S.Y., and Mittal, G.S. Electroporation of cell membranes: a review. Crit Rev Biotechnol 16, 349-62, 1996.

Inoue, T., and Krumlauf, R. (2001). An impulse to the brain-using in vivo electroporation. Nat Neurosci 4 Supp 1, 1156-1158, 2001.

Itasaki, N., Bel-Vialar, S., and Krumlauf, R. 'Shocking' developments in chick embryology: electroporation and in ovo gene expression. Nat Cell Biol 1, E203-207, 1999.

Kinosita, K., Jr., and Tsong, T.Y. Voltage-induced conductance in human erythrocyte membranes. Biochim Biophys Acta 554:479-497, 1979.

Koshiba-Takeuchi, K., Takeuchi, J.K., Matsumoto, K., Momose, T., Uno, K., Hoepker, V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K., and Ogura, T. Tbx5 and the retinotectum projection. Science 287:134-7, 2000.

Marszalek, P.E., Farrell, B., Verdugo, P., and Fernandez, J.M. Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin. Biophys J 73:1169-1183, 1997.

Miyasaka, N., Arimatsu, Y., and Takiguchihayashi, K. Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation. Neuroreport 10:2319-2323, 1999.

Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., and Yasuda, K. Efficient targeting of gene expression in chick embryos by microelectroporation. Dev Growth Differ 41:335-344, 1999.


References • 59

Muramatsu, T., Mizutani, Y., Ohmori, Y., and Okumura, J. Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo. Biochem Biophys Res Commun 230:376-380, 1997.

Muramatsu, T., Nakamura, A., and Park, H.M. In vivo electroporation: a powerful and convenient means of nonviral gene transfer to tissues of living animals (Review). Int J Mol Med 1, 55-62, 1998.

Neumann, E., Kakorin, S., Tsoneva, I., Nikolova, B., and Tomov, T. Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation. Biophys J 71: 868-77, 1996.

Neumann, E., Toensing, K., Kakorin, S., Budde, P., and Frey, J. Mechanism of electroporative dye uptake by mouse B cells. Biophys J 74:98-108, 1998.

Neumann, E., Kakorin, S., and Toensing, K. Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem Bioenerg 48:3-16, 1999.

Olofsson, J., Nolkrantz, K., Ryttsen, F., Lambie, B.A., Weber, S.G., and Orwar, O. Single-cell electroporation. Curr Opin Biotechnol 14:29-34, 2003.

Sakamoto, K., Nakamura, H., Takagi, M., Takeda, S., and Katsube, K. Ectopic expression of lunatic Fringe leads to downregulation of Serrate-1 in the developing chick neural tube; analysis using in ovo electroporation transfection technique. FEBS Lett 426:337-41, 1998.

Swartz, M., Eberhart, J., Mastick, G.S., and Krull, C.E. Sparking new frontiers: using in vivo electroporation for genetic manipulations. Dev Biol 233:13-21, 2001.

Teruel, M.N., Blanpied, T.A., Shen, K., Augustine, G.J., and Meyer, T. A versatile microporation technique for the transfection of cultured CNS neurons. J Neurosci Methods 93:37-48, 1999.

Weaver, J.C. Electroporation: a general phenomenon for manipulating cells and tissues. J Cell Biochem 51:426-435, 1993.

References

60 • References

Weaver, J.C. Electroporation theory. Concepts and mechanisms. Methods Mol Biol 55:3-28, 1995.

Yasuda, K., Momose, T., and Takahashi, Y. Applications of microelectroporation for studies of chick embryogenesis. Dev Growth Differ 42:203-206, 2000.

Yasugi, S., and Nakamura, H. Gene transfer into chicken embryos as an effective system of analysis in developmental biology. Dev Growth Differ 42:195-197, 2000.

Zheng, Q.A., and Chang, D.C. High-efficiency gene transfection by in situ electroporation of cultured cells. Biochim Biophys Acta 1088:104-110, 1191.


Technical Assistance • 61

Technical Assistance

If you need help to resolve a problem, there are several ways to contact Axon Instruments / Molecular Devices:

World Wide Web www.moldev.com

Phone 1 (800) 635-5577

Fax

E-mail www.moldev.com/support

Questions? See Axon's Knowledge Base: http://www.moldev.com/support

Technical Assistance

http://www.axon.com/

Warranty and Repair Service • 63

Warranty and Repair Service

Warranty Axon Instruments / Molecular Devices warrants its non-consumable hardware products to be free from defects in materials and workmanship for 12 months from date of invoice. The warranty covers the cost of parts and labor to repair the product. Products returned to our factory for repair must be properly packaged with transportation charges prepaid and the shipment fully insured. Axon Instruments / Molecular Devices will pay for the return shipping of the product to the customer. If the shipment is to a location outside the United States, the customer will be responsible for paying all duties, taxes and freight clearance charges if applicable.

The warranty is valid when the product is used for its intended purpose and does not cover products which have been modified without approval from Axon Instrument / Molecular Devices, or which have been damaged by abuse, accident or connection to incompatible equipment.

To obtain warranty service, follow the procedure described in the Repair Service section. Failure to do so will cause long delays and additional expense to the customer.

This warranty is in lieu of all other warranties, expressed or implied.


64 • Warranty and Repair Service

Repair Service The company reserves the right to cease providing repair maintenance, parts and technical support for its non-consumable hardware products five years after a product is discontinued. Technical support for old versions of software products will cease 12 months after they are upgraded or discontinued.

If you purchased your instrument from a Distributor or OEM Supplier, contact them for repair service.

If you purchased your instrument from Axon Instruments / Molecular Devices, contact our Technical Support Department. If it is determined your instrument must return to the factory for repair, the Technical Support Representative will issue a Service Request (SR) number. Our Logistic Coordinator will contact you with specific instructions.

Shipping The Axoporator 800A is a solidly built instrument designed to survive shipping around the world. However, in order to avoid damage during shipping, the Axoporator 800A must be properly packaged.

In general, the best way to package the Axoporator 800A is in the original factory carton. If this is no longer available, we recommend that you carefully wrap the Axoporator 800A in at least three inches (75 mm) of foam or "bubble-pack" sheeting. The wrapped instrument should then be placed in a sturdy cardboard carton. Mark the outside of the box with the word FRAGILE and an arrow showing which way is up.

We do NOT recommend using loose foam pellets to protect the Axoporator 800A. During shipping, there is a good chance that the instrument will shift within the loose pellet packing and be damaged.

If you need to ship the Axoporator 800A to another location, or back to the factory, and you do not have a means to adequately package it, Axon Instruments can ship


Warranty and Repair Service • 65

the proper packaging material to you for a small fee. This may seem an expense you would like to avoid, but it is inexpensive compared to the cost of repairing an instrument that has sustained shipping damage.

It is your responsibility to package the instrument properly before shipping. If the packaging is inadequate, and the instrument is damaged during shipping, the shipper will not honor your claim for compensation.


Declaration of Conformity • 67

Declaration of Conformity

Manufacturer: Axon Instruments / Molecular Devices 3280 Whipple Road Union City, CA 94587 USA Type of Equipment: Single-Cell Electroporator

Model Number: Axoporator 800A

Year of Manufacture: 2004

Application of Council Directives:

EC EMC Directive 89/336/EEC as amended

EC Low Voltage Directive 73/23/EEC as amended

Harmonized Standards to which Conformity is Declared:

EMC: EN 61326: 1998 (A2: 2001) EN 55011/AS/NZSCISPR11: 2002 (Group 1, Class B) Safety: EN 61010-1: 2001

I, the undersigned, hereby declare that the equipment specified above conforms to the above Directives and Standards.

Authorized Signature and Date: (Signature on file)

Declaration of Conformity

Important Safety Information • 69

Important Safety Information

DISCLAIMER

THIS EQUIPMENT IS NOT INTENDED TO BE USED AND SHOULD NOT BE USED IN HUMAN EXPERIMENTATION OR APPLIED TO HUMANS IN ANY WAY. THIS UNIT GENERATES HIGH VOLTAGE AND CARE SHOULD BE TAKEN IN ITS OPERATION IN ANY ENVIRONMENT.

WARNINGS MAIN UNIT AND HEADSTAGE

NO CUSTOMER SERVICEABLE COMPONENTS ARE CONTAINED WITHIN THIS UNIT. CONSULT THE MANUFACTURER FOR REPAIR/RETURN INSTRUCTIONS.

IF THE EQUIPMENT IS USED IN ANY MANNER NOT SPECIFIED BY THE MANUFACTURER, THE PROTECTION PROVIDED BY THE EQUIPMENT MAY BE IMPAIRED.

HAZARDOUS VOLTAGE MAIN UNIT

To prevent electric shock and damage to the instrument, do not attempt to open unit.

HAZARDOUS VOLTAGE HEADSTAGE

To prevent electric shock and damage to the instrument, do not attempt to open unit. The voltage and current present at the headstage Output connector and brass case connector may produce an electric shock. To avoid receiving an electrical shock from the headstage, do not make physical contact between the input for the micropipette holder and the signal ground input of the headstage when the Axoporator is switched ON. Circuitry contained within the main unit limits the voltage and current present at the headstage Output connector to voltages ≤ ±100 VDC and currents ≤2 mADC under normal operation and ≤ ±150 VDC and ≤15 mADC under single fault conditions. This device complies with IEC 61010: Measurement Category III.

POWER-SUPPLY Supply Voltage

The power supply of the Axoporator 800A can be directly connected to all international supply voltages. The input range is from 100 to 240 V~. No range switching is required.

Resetting Axoporator following power supply shut down

If the power supply shuts down, disconnect the power cord

from the outlet and disconnect the power supply from the Axoporator.

Investigate the reason for the circuit overload.

If it is deemed safe to do so, reconnect the power cord to the outlet and the power supply to the Axoporator.

Basic Equipment Setup and Safety

1. Connections: Use the included IEC power cord to connect the instrument to a GROUNDED power receptacle. That is, the mains power cord must be plugged into an earthed mains outlet. Routinely inspect the mains power cord and replace it if it’s damaged.

2. Assembly: The headstage connects to the instrument through the rear panel, 15 pin D-sub connector marked "Headstage". Power should always be turned OFF when the headstages is connected to or disconnected from the main unit.

3. There are no user serviceable parts. Do not open the case of the Axoporator, headstage or power supply. Contact the factory for repairs.

4. Cleaning: Remove the power cord from the unit. Use a soft cloth moistened with distilled water to clean dust and salt spills from the Axoporator 800A and AP-1A headstage. Do not use solvents or detergents. Do not use abrasive material. Avoid spilling liquids on the Axoportator 800A or AP-1A headstage.

The Teflon input connector should be kept very clean. Effective cleaning can be done by swabbing carefully with denatured ethyl or isopropyl alcohol or deionized water. If possible, avoid the use of Freon since it is thought to be detrimental to the environment.

Safe Environmental Conditions

1. Indoor use.

2. Mains supply fluctuations: not to exceed ±10% of the nominal voltage.

3. Temperature: between 5 ºC and 40 ºC.

4. Altitude: up to 2000 m.

5. Humidity: maximum relative humidity 80% for temperatures up to 31 ºC decreasing linearly to 50% relatitve humidity at 40 ºC.

6. Overvoltage: withstands a transient overvoltage impulse in accordance with category II of IEC 60363-4-443.

7. Pollution Degree: suitable for use in POLLUTION DEGREE 2 environment.

8. This instrument is designed to be used under laboratory conditions. Operate in a clean, dry environment only. Do not operate in a wet or damp environment.

Static Precautions

If you are in a laboratory where static is high (i.e., you hear and feel crackles when you touch things), you should touch a grounded metal object immediately before touching the headstage.

Important Safety Information

Index • 71

Index

AP-1A headstage, 5 Audio Monitor, 13. (See Headphone) Connections, 10

Headstage, 11 Power Supply, 10

Controls, 17 ABORT/RESET, 17 MANUAL COUNT, 17 MENU Button, 17 MENU Dial, 17 OFFSET VOLTAGE, 17 PULSE FREQUENCY, 17 PULSE TYPE, 17 PULSE VOLTAGE, 17 PULSE WIDTH, 17 SINGLE/TRAIN, 17 TRAIN DURATION, 17 TRIGGER, 17

Display, 18

Automatic Count, 19 Current, 19 Manual Count, 19 Menu Settings, 21 Offset Voltage, 19 Pipette Resistance, 19 Pulse and Train

Characteristics, 20

Time, 19 Total Power, 19 Voltage, 19

Headstage, 14

Bath Connection, 16 Cleaning, 16, 69 Mounting, 16 Noise, 16 Precautions

High voltage, 14 Hot plugging, 14 Static, 14

Types, 14 Voltage Clamp Circuit, 14

Inputs/Outputs, 22

EXT COMMAND, 23 EXT. TRIGGER, 22 FOOT SWITCH, 22 Headphone, 22 HEADSTAGE, 24 IOUTPUT, 23 SYNC OUTPUT, 22 VOUTPUT, 23

Micromanipulator, 9 Micropipette Holder, 28

Adapters, 31

Design, 28 Maintenance, 31 Use, 30

Micropipettes Fabricating, 33 Filling, 34 Mounting, 36 Positioning, 36

Microscope, 10 Model Cell, 32 Oscilloscope, 11 Protocols, 40

Bi-level pulses, 42 Rectangular pulses, 41

Pulses and Trains, 25 Terminology, 27 Triggering, 28 Using Axoporator's Pulse

Generator, 27 SCE-1U model cell, 5 Single-cell electroporation, 2 Stimulation Parameters, 38 Testing the Setup, 43

Index

axoporator 800a

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