Related Manuals for Warner OC-725C
Summary of Contents for Warner OC-725C
- Page 1 Oocyte Clamp Amplifier User’s Manual OC-725C Warner Instruments Publication 5720-001-REV-1.0...
- Page 2 Table of Contents SUBJECT PAGE # Warranty Safety Introduction Unique Features High Voltage Compliance Bath Clamp Headstage Dual Oocyte Studies Voltage Headstage Probe Voltage and Current Meters Additional Features Buzz controls Electrode Test Capacity Compensation Overload Alarm DC Offsets Nomenclature Text conventions Control Description 9-15...
- 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 Single oocyte setup with indirect ground Single oocyte setup with direct ground Dual ooctye set-up...
- Page 4 Table of Contents SUBJECT PAGE # Comments and Recommendations 32-33 Membrane damage Repeated recordings Electrophysiology Appendix 34-37 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...
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Page 5: Warranty
Warranty Warner Instruments warranties this instrument for a period of one year from date of purchase. At its option, Warner Instruments will repair or replace the unit if it is found to be defective as to workmanship or material. This warranty does not extend to damage resulting from misuse, neglect or abuse, normal wear and tear, or accident. -
Page 6: Safety
Use Proper Line Cord Use only a line cord that is certified for country of use. The Operating voltage range for the OC-725C is 100-120~, or 200-240v~. Ground the Product This product is grounded through the grounding conductor of the power cord. To avoid elextric shock, the grounding conductor must be connected to earth ground. -
Page 7: Introduction
The instrument has several features making it ideal for these purposes. Unique Features High Voltage Compliance: The OC-725C combines high AC and DC gains and a voltage compliance of ±180 V to insure fast, nonsaturating clamp performance under nearly any condition. -
Page 8: Introduction
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. Capacity Compensation for the V voltage input. 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. -
Page 9: Nomenclature
Since our goal is to provide clarity rather than complexity, we welcome any feedback you may wish to provide. • Warner Instrument product numbers are presented using a bold type. • References to instrument panel control blocks are specified using underlined small caps. -
Page 10: Control Description
The V x10 output BNC reports the membrane voltage in mV multiplied by 10. negative capacity compensation (-C) has been added to the OC-725C allowing for its use as an electrometer in intracellular measurements. Input capacitance up to 45 pF can be neutralized using the two associated controls. -
Page 11: Bath Electrodes
Control Description In general, negative capacity compensation is not useful for oocyte clamp applications since clamp speed is a function of (1) the current electrode resistance, (2) the RC time constant of the oocyte (typically 1 MΩ in parallel with 0.5 µF), and (3) the compliance voltage of the clamp current. -
Page 12: Clamp Section
Control Description Current outputs are available from the I MONITOR BNC at full bandwidth (~10 kHz) and from the I MONITOR FILTERED BNC which is filtered at 1 kHz by an integral 4-pole Bessel filter. Clamp Section The CLAMP control block contains the MODE SELECTOR switch as well as the GAIN and DC GAIN controls. -
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 the digital potentiometer thumbwheel and range toggle switch. Ranges are ±99 mV and ±198 mV depending on the scale multiplier selected (x1.0 or x2.0). -
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 and GAIN TELEGRAPH output BNCs, the ALARM switch and instrument GROUNDS. -
Page 15: Bath Headstage
Comments Connecting to Line Power The model OC-725C is supplied with a 3-conductor power cord. One conductor provides a connection between the instrument housing and the earth ground. Safe operation of the instrument will be assured provided that the ground circuit in the power outlet is wired correctly and is connected to earth. -
Page 16: Control Description
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
Initial Instrument Settings Connect the model cell to the OC-725C as shown on its cover. Be sure to connect the ground wire to the ground mini-jack on the side of the bath probe. Connect the Vm x10 BNC and the I MONITOR BNC on the OC-725C to an oscilloscope. -
Page 18: Test Procedures
Using the Model Membrance 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 Membrance 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. A1kHz 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 Membrance 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: Single Oocyte Setup With Direct Ground
Setup Single Oocyte Setup with Direct Ground Applications where use of the bath clamp is not suitable (such as those with a very long solution path to ground) can be configured using the alternate method of directly grounding the bath as shown in Figure B. In this configuration, current is read from the “high side”... -
Page 25: 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 26: 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 1 V/µA i monitor output bath electrode gain select bath electrode 0 mV hold commands polarity toggle commands mode select clamp clamp dc gain toggle clamp... -
Page 27: Setup
Setup Gain Headstage output max output Maximum Select Resistor range (V/µA) (µA) meter reading x0.1 10 kΩ 0.01 - 1.0 10 - 1000 199.9 µA x1.0 100 kΩ 0.1 - 10 1 - 100 19.99 µA 1 MΩ 1.0 - 100 0.1 - 10 1.999 µA Other Gain Range Selection Considerations... -
Page 28: 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 29: 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 30: 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 31: A Procedure For Recording From Oocytes
A Procedure for Recording from Oocytes Unclamping the Cell 19) To unclamp the cell, turn the GAIN control (CLAMP section) fully counter- clockwise to the detent off position. This will also disengage the DC GAIN. NOTE: If the control is not fully off and the DC GAIN is left on, the preparation will not be unclamped. -
Page 32: Special Circumstances
This problem is overcome by using the optional DIFFERENTIAL VOLTAGE HEADSTAGE. Configuration The OC-725C current measuring circuit can be changed to the high side current measuring mode by setting a dip switch on the main circuit board. 1) First disconnect the power cord from the wall. -
Page 33: 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 34: Comments And Recommendations
NOTE: Specialized equipment suitable for oocyte studies (oocyte chambers, perfusion systems, a Faraday cage, a noise-free DC lamp, and micro-manipulators) are available from Warner Instruments. Although it is not required, a computer can be employed to control the command voltage. Acquisition and display of data is also usually handled by computer. The OC- 725C is fully compatible with all commercially available software packages designed for electrophysiological research. -
Page 35: 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 Negative Capacity 0 - 45 pF V m Meter Range, full scale... - Page 36 Appendix SPECIFICATIONS Current electrode channel (V e ) Compliance Voltage Alarm ± 160 V Gain Variable 0 - 2000 AC/DC 1 x106 DC, switch selected Electrode Test 10 mV/MΩ Commands Hold (internal) ± 198 mV in 2 ranges External input 1 V in = 0.1 V command (attenuated by 10) Maximum external input...
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Page 37: Gain Select Settings
Appendix Gain Telegraph Outputs Output (V/µA) Gain 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 2.0 V 2.2 V 2.4 V 2.6 V Gain Select Settings Gain Headstage Output... -
Page 38: Appendix
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 39 Notes Publication 5720-001 REV1.0...
- Page 40 Notes www.warneronline.com...
- Page 41 Notes Publication 5720-001 REV1.0...
- Page 43 U.S.A. Sweden Harvard Apparatus CMA Microdialysis AB 84 October Hill Road Torshamnsgatan 30A Holliston, Massachusetts 01746 SE-164 40 KISTA, Sweden Phone (508) 893-8999 Phone +46.8.470.10.00 Toll Free (800) 272-2775 E-mail cma@microdialysis.se (508) 429-5732 www.microdialysis.com E-mail support@hbiosci.com www.harvardapparatus.com Spain Panlab S.L.U. Canada C/ Energia, 112 Harvard Apparatus, Canada...
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