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Axon Axoporator 800A Theory And Operation

Single-cell electroporator
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800.998.MATE
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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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Axoporator 800A
SINGLE-CELL ELECTROPORATOR
Theory and Operation
Part Number 2500-0158 Rev C March 2005 Printed in USA
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650 University Ave #5, Berkeley, CA 94710 USA
5 1 0 . 6 6 5 . 3 9 7 5
|
e - m a i l
i n f o @ a u t o m 8 . c o m

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Summary of Contents for Axon Axoporator 800A

  • Page 1 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.
  • Page 3 !!!!! SAFETY LEASE READ 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.

  • Page 5: Table Of Contents

    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.....................
  • Page 6 ..............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.
  • Page 7 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 ..................

  • Page 9: Chapter 1 Introduction

    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;...
  • Page 10 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.
  • Page 11 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.

  • Page 13: Chapter 2 Functional Checkout

    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...
  • Page 14 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.
  • Page 15 Functional Checkout • 7 Figure 1. Connections between the AP-1A headstage to the SCE-1U model cell. Functional Checkout...

  • Page 17: Chapter 3 Installation

    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.

  • Page 18: Microscope

    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.

  • Page 19: Optional Configurations

    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.

  • Page 21: Chapter 4 Reference Section

    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.

  • Page 22: Headstage

    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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 23 Reference Section • 15 Figure 2. Schematic of single cell electroporation. R =feedback resistance, R =electrode resistance, R =cleft resistance. V =command voltage, V =voltage at surface of membrane. Vo = output of amplifier A1. Vo =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 R and R...

  • Page 24: Noise

    Wait about ten minutes and insert a fine wick of cotton into the socket to dry the input socket. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 25: Front Panel Controls

    Reference Section • 17 Front Panel Controls Six push buttons and six knobs comprise the front panel controls. 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.

  • Page 26: Front Panel Display

    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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 27 Reference Section • 19 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 –...
  • Page 28 In addition, when trains are selected, the train duration and pulse frequency must be set. out-of-range Pulse frequency, pulse width and train duration are all interrelated. If an setting is made, the value selected will dim on the display. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 29 Reference Section • 21 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.

  • Page 30: Rear Panel Inputs/Outputs

    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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 31 Reference Section • 23 Figure 8. Inputs/Outputs, bottom row. : Electrode voltage (Vo) output. Provides a 0.1V signal for 1V delivered OUTPUT 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.
  • Page 32 24 • Reference Section HEADSTAGE: Connector for AP-1A headstage. For Factory Test Only: For factory test procedures. : Connector for external power supply. 12-14 V On/Off switch Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 33: Overview Of Pulses And Trains

    Reference Section • 25 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.
  • Page 34 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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 35: Terminology

    Reference Section • 27 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 ∗...

  • Page 36: Triggering

    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 Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 37 (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 headstages and is secured in place with a threaded collar.

  • Page 38: Holder Use

    The chlorided tip of the wire should be inserted into this solution. Avoid wetting the holder since this will increase the noise. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 39: Holder Maintenance

    Reference Section • 31 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 wn to the bare metal using fine sand paper and immerse the cleaned wire in bleach for...

  • Page 40: Model Cell

    2 mm gold socket on the model cell connects to the 2 mm grounding socket on rear of the AP-1A headstage. The circuit is shown in Figure 12. Two resistance settings, 10 MΩ and 15 MΩ, are available. Figure 12. SCE-1U circuit. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 41: Chapter 5 Tutorial

    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.
  • Page 42 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 Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 43 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.
  • Page 44 It remains to be Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 45 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.

  • Page 46: Selecting The Stimulation Parameters

    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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 47 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.

  • Page 48: Overview Of Protocols For Loading Dissociated Cells In Culture

    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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 49 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.
  • Page 50 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, Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 51: Testing The Setup

    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’...
  • Page 52 Determine which pulse parameters provide best control over dye movement. Optimal parameters should be able to deliver concise boluses of dye with no leakage. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 53: Chapter 6 Troubleshooting Guide

    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.

  • Page 54: Symptom: Positional Drift

    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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 55: Symptom: Current Or Power Reading Is Out Of Range

    Duty cycle requested is > 50%. Suggestion: Reduce pulse frequency or width. Symptom: No R value is reported Possible cause: Headstage is not properly connected. Suggestion: Switch off the Axoporator 800A and check the connection to the headstage input. Troubleshooting Guide...

  • Page 57: Chapter 7 Specifications

    Specifications • 49 Chapter 7 Specifications Unless otherwise specified, T A = 20 º C, 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.)

  • Page 58: Ap-1A-1Mu (Standard)

    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 Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 59: Micropipette Holder

    Specifications • 51 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.

  • Page 60: Offset Voltage

    (‘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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 61: Lcd Display

    Specifications • 53 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.

  • Page 62: Rear Panel

    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 Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 63 Specifications • 55 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...

  • Page 64: Power Requirements (Using The Provided Power Supply)

    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 Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 65: References

    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.
  • Page 66 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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
  • Page 67 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).
  • Page 68 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. Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.

  • Page 69: Technical Assistance

    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.axon.com Phone 1 (800) 635-5577 +1 (510) 675-6300 E-mail axontech@axon.com Questions? See Axon's Knowledge Base: http://support.axon.com...

  • Page 71: Warranty And Repair Service

    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.
  • Page 72 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...
  • Page 73 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.

  • Page 75: Declaration Of Conformity

    Declaration of Conformity • 67 Declaration of Conformity Manufacturer: Axon Instruments / Molecular Devices 3280 Whipple Road Union City, CA 94587 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...

  • Page 77: Important Safety Information

    To prevent electric shock and damage to the instrument, do not from the Axoporator 800A and AP-1A headstage. Do not use attempt to open unit. solvents or detergents. Do not use abrasive material. Avoid HAZARDOUS VOLTAGE HEADSTAGE spilling liquids on the Axoportator 800A or AP-1A headstage.

  • Page 79: Index

    Index • 71 Index AP-1A headstage, 5 Time, 19 Design, 28 Audio Monitor, 13. (See Headphone) Total Power, 19 Maintenance, 31 Voltage, 19 Use, 30 Connections, 10 Micropipettes Headstage, 11 Headstage, 14 Fabricating, 33 Power Supply, 10 Bath Connection, 16 Filling, 34 Controls, 17 Cleaning, 16, 69...

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