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Harvard Bioscience Warner OC-725D User Manual

Oocyte clamp amplifier
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Oocyte Clamp
Amplifier
User's Manual
OC-725D
Warner Instruments
Publication 5720-001-REV 1.2

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Summary of Contents for Harvard Bioscience Warner OC-725D

  • Page 1 Oocyte Clamp Amplifier User’s Manual OC-725D Warner Instruments Publication 5720-001-REV 1.2...

  • Page 2: Table Of Contents

    Table of Contents SUBJECT PAGE # Warranty Safety Introduction 6–7 Unique Features High Voltage Compliance Bath Clamp Headstage Voltage Headstage Probe Voltage and Current Meters Additional Features Buzz controls Electrode Test Overload Alarm DC Offsets Nomenclature Text conventions Control Description 9–15 Front panel Voltage electrode...
  • Page 3 Table of Contents SUBJECT PAGE # Initial instrument settings Test procedures Offset controls Voltage electrode test Buzz Current electrode test DC clamp test AC clamp test Setup 20–26 Pipettes Electrode holders Bath probe Electrode placement and grounding Bath clamp electrode placement Cable connections Resting position of the controls Using the gain select...
  • Page 4 Table of Contents SUBJECT PAGE # Appendix 32–35 Specifications — Equipment is intended to be operated in a controlled laboratory environment Gain telegraph outputs Gain select settings Noise from bath clamp feedback resistor References www.warneronline.com...

  • Page 5: Warranty

    Warranty & Repair Information Serial Numbers All inquires concerning our product should refer to the serial number of the unit. Serial numbers are located on the rear of the chassis. Calibrations All electrical apparatus is calibrated at rated voltage and frequency. Warranty Warner Instruments warranties this instrument for a period of one year from date of purchase.

  • Page 6: Safety

    Safety Information Please read the following safety precautions to ensure proper use of your valve controller. If the equipment is used in a manner not specified, the protection provided by the equipment may be impaired. To Prevent Hazard or Injury Use Proper Line Cord Use only a line cord that is certified for country of use.

  • Page 7: Introduction

    Introduction The model OC-725D Oocyte clamp is designed for two-electrode, whole-cell voltage clamping of Xenopus oocytes, as well as for other large cells and cell structures such as squid axons. The instrument has several features making it ideal for these purposes. Unique Features High Voltage Compliance: The OC-725D combines high AC and DC gains and a voltage compliance of ±180 V to insure fast, nonsaturating clamp performance under nearly any...

  • Page 8: Additional Features

    Introduction Additional Features Buzz controls for each electrode aid in penetration of cell membranes with a minimum of leakage. Electrode Test for voltage and current electrodes. Overload Alarm serves as a reminder when the feedback amplifier reaches its maximum output voltage, a condition which could result in damage to the oocyte. DC Offsets for voltage and current electrodes.

  • Page 9: Nomenclature

    Nomenclature Text Conventions This manual refers to amplifier controls at four functional levels; operational sections, control blocks, specific controls within a block, and settings of specific controls. To minimize the potential for confusion, we have employed several text conventions which are specified below.

  • Page 10: Control Description

    Control Description The OC-725D is comprised of three functional channels: A high impedance voltage sensing channel with input offset to measure membrane potential, a current sensing channel with bath clamp to clamp the bath and measure the membrane current, and a high voltage amplifier to deliver the clamping current.

  • Page 11: Bath Electrodes

    Control Description Bath Electrodes The bath electrodes control block contains the bath probe connector, the current meter, the gain select and FILTER SELECT switches, and the i monitor output BNC’s. The current meter reads the voltage (V ) of the current electrode when the clamp mode selector switch is off (see clamp section).

  • Page 12: Clamp Section

    Control Description Current output is available from the I MONITOR BNC at the bandwidth set by the FILTER switch. Clamp Section The CLAMP control block contains the MODE SELECTOR switch as well as the GAIN and DC GAIN controls. The CLAMP MODE SELECTOR switch selects for slow and fast clamp speeds, or for off.

  • Page 13: Commands

    Control Description Commands The commands control block contains the hold controls and command in÷10 input BNC. hold controls - hold potential is set with a ten turn potentiometer and RANGE toggle switch. Ranges are 0- +/-100 and 0-+/- 200mV depending on the scale multiplier selected (x10 or x20).

  • Page 14: Rear Panel

    Control Description Rear Panel The line power connector and fuse are located on the rear panel. Operating voltage is specified on the MODEL/SERIAL NUMBER sticker applied to the rear of the instrument. The rear panel also contains Ve x10, GAIN and FILTER TELEGRAPH output BNC's, the ALARM switch and instrument GROUNDS..

  • Page 15: Bath Headstage

    Control Description Bath Headstage The BATH PROBE is housed in a 2.8 x 3.5 x 4.2 cm aluminum enclosure. Inputs are two 1 mm pin jacks labeled I SENSE and I OUT. The case is electrically grounded and a pin jack is located on the side for connecting to shields.

  • Page 16: High Voltage Outputs

    Control Description NOTE: If the ground pin of the power cord is removed for any reason the instrument chassis must be directly connected to earth ground High Voltage Outputs When handling the current electrode cable, be sure to set the GAIN CONTROL fully clockwise and the CLAMP MODE switch to off.

  • Page 17: Using The Model Membrane

    Using the Model Membrane The model cell supplied with the OC-725D can be used for two purposes. First, novice users will find it a convenient tool for gaining experience in the operation of the instrument. Additionally, it is a convenient tool for trouble shooting since the function of the instrument can be quickly checked.

  • Page 18: Test Procedures

    Using the Model Membrane Test Procedures In the following testing procedures, allow a tolerance of ±1% on the readings taken. For example, if the test response is indicated as 100 mV, a reading from 99.0 to 101.0 mV would be within tolerance. Offset Controls Vm OFFSET (VOLTAGE ELECTRODE section): The full range of this control is ±200 mV.

  • Page 19: Buzz

    Using the Model Membrane Buzz This test is performed using controls in the VOLTAGE ELECTRODE section. Set the oscilloscope sensitivity to 5 V/div and depress the BUZZ pushbutton while monitoring the Vm x10 output. A 1KHz square wave of approximately 24 V p-p will be generated as long as the button is depressed.

  • Page 20: Ac Clamp Test

    Using the Model Membrane AC Clamp Test Adjust the Vm OFFSET control to 0.0 V. Apply a 0.8 V, 100 Hz square wave to the COMMAND IN ÷10 BNC. Monitor the Vm x10 and I MONITOR outputs on the oscilloscope. Switch the CLAMP MODE switch to fast and increase the GAIN until Vm reads 80 mV.

  • Page 21: Setup

    Setup The following instructions are designed to guide the user, step-by-step, through a typical recording session involving a Xenopus oocyte. It is assumed that the user is already familiar with the techniques of Xenopus oocyte excision and microinjection (for a review of those techniques, see Colman, 1984).

  • Page 22: Electrode Holders

    Setup Electrode Holders Voltage Electrode — The voltage recording electrode holder uses a silver wire for the electrical coupling between the pipette and holder connector. Any silver wire contacting the KCl solution in the pipette must be chlorided to reduce junction potentials (see Chloriding Procedure in Appendix).

  • Page 23: Bath Clamp Electrode Placement

    Setup Bath Clamp Electrode Placement Proper placement of the bath electrodes (Iout and Isense) is important for obtaining optimum performance. The Isense electrode (or the agar bridge associated with it) should be placed as close to the oocyte as possible since this point is the virtual ground node, and on the same side as the voltage recording electrode.

  • Page 24: Cable Connections

    Setup Cable Connections Bath clamp headstage: After positioning the probe as described above, connect it to the BATH PROBE socket (BATH ELECTRODES section). Voltage electrode headstage: The high impedance probe for recording membrane potential should be mounted on a micro-manipulator and connected to the VOLTAGE PROBE socket (VOLTAGE ELECTRODE section).

  • Page 25: Resting Position Of The Controls

    Setup Resting Position of the Controls To begin, set the instrument controls to the following positions: Control Section Setting power i monitor output bath electrode 1 V/µA gain select bath electrode hold commands 0 mV polarity toggle commands mode select clamp clamp dc gain toggle clamp...

  • Page 26: Other Gain Range Selection Considerations

    Setup Gain Headstage output max output Maximum Select Resistor range (V/µA) (µA) meter reading x0.1 10 kΩ 0.01 - 2.0 5 - 1000 199.9 µA x1.0 100 kΩ 0.1 - 20 0.5 - 100 19.99 µA 1 MΩ 1.0 - 200 0.05 - 10 1.999 µA Other Gain Range Selection Considerations...

  • Page 27: A Procedure For Recording From Oocytes

    A Procedure for Recording from Oocytes Initial Electrode Placement 1) Make sure that the bath electrodes are submerged in the chamber (or in the agar bridge wells with the agar bridges completing the circuit to the bath) and the oocyte is stable on the chamber floor. 2) Install the voltage and current pipettes onto their respective holders but do not yet place them in the chamber bath solution.

  • Page 28: Current Electrode Placement

    A Procedure for Recording from Oocytes Current Electrode Placement Advance the current electrode until the tip is in the chamber bath solution. Adjust V OFFSET for a zero reading on the CURRENT ELECTRODE METER. This will establish a null reference allowing the resting potential to be directly read.

  • Page 29: Clamping The Cell

    A Procedure for Recording from Oocytes Now, advance the current electrode until its tip is slightly depressing the plasma membrane of the cell and depress the Ve BUZZ pushbutton. Similar to the voltage electrode BUZZ, the current electrode BUZZ produces a 1 V, 1 kHz oscillation across the current electrode.

  • Page 30: Unclamping The Cell

    A Procedure for Recording from Oocytes Unclamping the Cell 19) To unclamp the cell, turn the GAIN control (CLAMP section) fully counter- clockwise. NOTE: If the control is not fully off and the DC GAIN is left on, the preparation will not be unclamped.

  • Page 31: Comments And Recommendations

    Comments and Recommendations Membrane Damage Recording from the same cell at a later time requires that the cell remain healthy during the interim incubation. The less damage done to the membrane during handling and impaling the cell, the happier it will be. Use of the BUZZ function should help minimize the trauma from electrode penetration.
  • Page 32 Comments and Recommendations In addition to mechanical isolation, the setup must also be isolated from external electrical noise sources. These include motors, lamps, and wiring. The platform should be shielded from these sources of electrostatic radiation with a Faraday cage. All equipment within the Faraday cage should be grounded to the rear panel instrument circuit ground and is best achieved by connecting everything (including the cage) to a ground bus within the cage.

  • Page 33: Appendix

    Appendix SPECIFICATIONS Voltage recording channel (Vm) Input Impedance 5 x1011 Ω, shunted by 3 pF Output Resistance 100 Ω V m OFFSET ± 200 mV at V probe input Noise* 50 mV RMS at 1 kHz Electrode Test 10 mV/MΩ V m Meter Range, full scale ±...
  • Page 34 Appendix SPECIFICATIONS Current electrode channel (V e ) Compliance Voltage ± 180 V Alarm ± 160 V Gain Variable 0 - 2000 AC/DC 1 x106 DC, switch selected Electrode Test 10 mV/MΩ Commands Hold (internal) ± 200 mV in 2 ranges External input 1 V in = 0.1 V command (attenuated by 10)

  • Page 35: Gain Telegraph Outputs

    Appendix Gain Telegraph Outputs Gain Telegraph Outputs Output (V/µA) Gain Telegraph Frequency (Hz) Filter Telegraph 0.01 0.2 V 0.02 0.4 V 0.05 0.6 V 0.8 V 1.0 V 1.2 V 1.4 V 1.6 V 1.8 V Bypass 2.0 V 2.2 V 2.4 V 2.6 V 2.8V...

  • Page 36: References

    Appendix References Colman, A. (1984). Translation of eukaryotic messenger RNA in Xenopus oocytes. Transcription and Translation, eds. B.D. Hames and S.J. Higgins (IRL Press, Oxford). Ch. 10 Hille, B. (1984). Ionic Channels of Excitable Membranes. Sinauer (Sunderland, MA). Ch. 2. Zhou, J., Potts, J.F., Trimmer, J.S., Agnew, W.S.
  • Page 37 Notes Publication 5720-001-REV 1.2...
  • Page 38 1125 Dixwell Avenue Hamden, CT 06514, USA Phone: 833.668.8632 Email: us-sales@smart-ephys.com Web: www.warneronline.com Find global support for Warner Instruments and all of the Smart Ephys brands at www.smart-ephys.com...

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