OC-725C User`s Manual (Rev. 050816.2)
Contents
1. The resulting voltage in mV will be displayed on the METER in the BATH ELECTRODE section From that value the resistance of the pipette can be calculated exactly as described above 1 e divide the readout by 10 to get the resistance in MQ Since the current electrode has a larger diameter its resistance should be less than that of the voltage electrode about 1 0 MQ or less If no voltage display is present during the electrode test procedure and you re sure that the electrode is contacting the bath perform the following checks a Make sure that all cables are connected properly b Check to see that the aluminum shield around the current electrode pipette if used is not touching the bath solution or the electrode wire c Check the current electrode to see if there is a bubble in the pipette causing an open circuit Impaling the cell 10 11 12 13 Recheck the VOLTAGE ELECTRODE METER to verify that the potential is correctly offset to read 0 0 mV and readjust the Vm OFFSET control if needed Advance the VOLTAGE ELECTRODE until its tip is slightly depressing the plasma membrane of the cell and depress the Vm BUZZ pushbutton This will produce a 1 V 10 kHz oscillation at the voltage electrode disrupting the membrane and causing the tip of the electrode to impale the cell with no further movement of the micro manipulator this technique will work best with fresh oocytes i e 1 or 2 days post excisi2. the following measurements were made with a shorted input with the standard 0 5 uF capacitance model cell and with a 0 22 uF capacitance modified model cell All readings were recorded at 1 kHz 8 pole Bessel and are RMS Noise Feedback Resistor Shorted Input Standard Model Cell Modified Model Cell 0 5 uF 0 22 uF 10 kQ 75 pA 6 0 nA 4 4nA 100 kQ 28 pA 5 5 nA 4 4nA 1 MQ 22 pA 5 0 nA 4 0 nA A lower value feedback resistor increases the speed of the bath clamp and can handle larger currents without saturating important when recording from high expression oocytes Warner instruments A Harvard Apparatus Company A PROCEDURE FOR RECORDING FROM OOCYTES Initial electrode placement 1 2 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 Install the voltage and current pipettes onto their respective holders but do not yet place them in the chamber bath solution Voltage electrode placement 3 4 5 6 Advance the voltage recording electrode into the bath The VOLTAGE ELECTRODE METER will indicate in mV the potential between the electrode and the bath If there is no voltage reading and you are sure that the pipette tip is in the bath solution perform the following checks a make sure that all cables are connected properly
3. 7 switches are off 4 To convert to high side current measuring mode turn S4 off and turn 82 S5 and S7 on All other switches should remain in the off position Optional voltage headstage The optional DIFFERENTIAL INPUT VOLTAGE RECORDING HEADSTAGE Model 7255DI is used in applications where the bath clamp headstage cannot be effectively employed Two examples are 1 situations where the solution path from oocyte to ground is very long and 2 when recording from two oocytes in a common bath Two 1 mm input jacks are located on the side of the headstage V DIFF and GND A shorting jumper is supplied and is used for normal single ended recording For double Warner Instruments A Harvard Apparatus Company ended recording the jumper is removed and a V DIFF ELECTRODE is connected to the V DIFF jack as shown below If a shield between the voltage and clamp current electrodes is used it should be connected to the headstage ground Warner Instruments A Harvard Apparatus Company 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 Membrane damage can be further reduced by properly isolating the experimental platform from vibratio
4. CLAMP MODE switch is set to off Return all controls to their initial settings when done with this test Voltage electrode test In actual practice the voltage electrode test is used prior to entering the cell and indicates the resistance of the electrode When used in conjunction with the model cell it measures both the electrode and membrane resistance 2 MQ This test is performed using controls in the VOLTAGE ELECTRODE section With the model cell in place depress the Vm ELECRODE TEST pushbutton and observe a reading of 20 mV on the VOLTAGE ELECTRODE METER This corresponds to a 2 MQ reading at a calibrated response of 10 mV MQ On the oscilloscope the Vm x10 output will read 200 mV which is x10 the applied test voltage Since the test current is being passed through 1 MQ Rn the I MONITOR output will indicate 1 mV which corresponds to 10 nA of current With the CLAMP MODE switch set to off the BATH ELECTRODE METER monitors Ve voltage at the current electrode In this case Ve will be a measure of the voltage across Rm and the meter will indicate 10 mV 1 MQ The Ve x10 output BNC on the rear panel can also be checked to see that it reads 100 mV meter reading x10 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 10 kHz square wave of approximately 24 V p p will be gen
5. off AC clamp test 100 Adjust the Vm OFFSET control to 0 0 V ei Apply a 0 8 V 100 Hz square wave to 40 the COMMAND IN 10 BNC Monitor the odi Vm x10 and I MONITOR outputs on the F Pi oscilloscope 40 4 Switch the CLAMP MODE switch to fast and increase the GAIN until Vm reads 80 FO 2 4 6 8 10 12 14 mV Verify that the Vm x10 BNC ms reports 0 8 V As you further increase 10 the cam control you will see the rise time of the oscilloscope trace become 4 faster since the speed of the clamp is 2 limited by the resistance of the gt current electrode and the capacitance 4 of the oocyte If ringing oscillation is u observed decrease the cam setting to obtain the fastest clean waveform as 0 2 4 6 8 10 12 14 ms Warner Instruments A Harvard Apparatus Company shown on the previous page The current signal I MONITOR shown in the figure displays the high current spikes required to charge the oocyte capacitance Set the HOLD control to a reading of 50 mV and switch the POLARITY toggle to pos The square wave will be displaced 50 mV in the positive direction Switching the polarity to neg will produce a 50 mV offset Warner Instruments A Harvard Apparatus Company 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
6. rather than complexity we welcome any feedback you may wish to provide gt Warner Instrument product numbers are presented using a bold type gt References to instrument panel control blocks are specified using UNDERLINED SMALL CAPS e g COMMANDS CLAMP gt References to specific controls within a block are specified using NON UNDERLINED SMALL CAPS e g MODE SWITCH DC GAIN Finally references to individual control settings are specified in italic type e g slow fast 100 mV gt Special comments and warnings are presented in highlighted text Any other formatting should be apparent from context Warner Instruments A Harvard Apparatus Company CONTROL DESCRIPTION The OC 725C is comprised of three functional channels A high impedance voltage sensing channel with capacity compensation and 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 The complete voltage clamp system consists of the OC 725C the voltage recording probe with electrode holder the current sensing bath probe with silver wire electrodes and the current cable with electrode holder Front panel The instrument front panel is divided into six control blocks titled VOLTAGE ELECTRODE Vm BATH ELECTRODES Im CLAMP COMMANDS and CURRENT ELECTRODE The instrument rear panel has BNC connec
7. the OC 725C 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 A schematic of the model cell is shown to the right The oocyte is represented by a 1 MO resistor shunted by a 0 47 uF capacitor The voltage 1M t 1M and current electrodes are i each represented by 1 MO VA n resistances and the bath to voltage to current probe cable probes are represented by the 10 kO resistors Warner Instruments A Harvard Apparatus Company 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 Set the instrument panel controls as follows and turn the POWER SWITCH on Control Control block Setting POWER SWITCH off Vm OFFSET VOLTAGE ELECTRODE Center of rotation approximately 5 turns OUTPUT GAIN BATH ELECTRODES 0 1 V uA GAIN SELECT BATH ELECTRODES x1 0 CLAMP MODE switch CLAMP off DC GAIN toggle CLAMP out GAIN CLAMP CCW to detent off HOLD POTENTIAL COMMANDS 00 mV HOLD POTENTIAL MULTIPLIER COMMANDS x1 0 HOLD POLARITY COMMANDS off Ve OFFSET CURRENT ELECTRODE Center of rotation approximate
8. 2 EN61000 4 2 EN61000 4 3 ENV50204 EN610000 4 4 EN610000 4 8 EN610000 4 11 Manufacturer s Name Warner Instruments LLC Manufacturer s Address 1125 Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Equipment Description Instrument Amplifier Equipment Class ITE Class A Model Numbers OC 725C I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Connecticut USA Pene Full Name Burton J Warner Signature Position President Warner Instruments A Harvard Apparatus Company Declaration of Conformity CE MARKING LVD Application of Council Directive 73 23 EEC Standards To Which Conformity Is EN61010 1 1993 Declared Manufacturer s Name Warner Instruments LLC Manufacturer s Address 1125 Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Equipment Description Instrument Amplifier Safety requirements for electrical equipment for measurement and laboratory use Equipment Class Class I Model Numbers OC 725C I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Connecticut USA Signature Gab pr Full Name Burton J Warner Position President Warner Instruments A Harvard Apparatus Company OC 725C Manual Rev 050816 2 33 WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Custo
9. OC 725C Manual Rev 050816 2 Warner Instruments Oocyte Clamp Amplifier Model OC 725C Warner Instruments 1125 Dixwell Avenue Hamden CT 06514 800 599 4203 203 776 0664 203 776 1278 fax CE OC 725C Manual Rev 050816 2 Table of Contents NOMENCEATURE DC 4 Text conventions EA AN A 4 CONTROL DESCRIPTION 2 005058 t 5 Front panel NN RN 5 Voltage electrode ete ee ee tete e ete Dee Ne ER ka Pu n cu a eee Lene eee Eee ere Rana Po heed a 5 Bath electrodes utis A HH RS e etta eis 6 Clamp sectioniz a siio e tor RE ee os ie eee e heben ert eee eade Saat ERE AEDs dn 6 C mman sa uincelenaichesneikunnnesheininsiehnhsstsehnninsersnhshtaesnnineiehehentnernshneissnhnhtehsnnineiehnhnsl enenkl kreshesitrahsthe 7 Current electrode rte ee e etna Forsitan ei a vedi a aia 7 horiWrud e 8 EDDIE FED HIDE ETE EL 9 Voltage recording headstages eese esee eene enne ettet entente setis entente enne 9 Bath headstage AERA S Re dee ex 9 Currentelectrode c ble ii nennen ana decanos lees addin rE Lada PATER RAE i P S 9 Model Cell ee iei ettet ottenere tti o Ea a Eee do LEER b E rne ode bord ea ed doceo PU LER De Leod 10 LICET E 10 Connecting to line power eee eei eet n e D E Ree Se ee e eg Bae ibe een 10 High voltage ouputs us etui tette IR c A be Pa eee e e daa Pe leas e ele lern ru eee 10 USING THE MODEL MEMBRANE ee e
10. and microinjection for a review of those techniques see Colman 1984 It is also assumed that the user has some familiarity with the basic circuitry of a two electrode voltage clamp for review see Hille 1984 Pipettes Microelectrodes can be made using the same glass tubing and dimensions as those used for a typical patch pipette and are usually filled with 3 M sterile filtered KCl Unlike the pipettes used as patching electrodes microelectrode pipettes do not require fire polishing nor coating with Sylgard They will need to be broken off however to a relatively large diameter to insure a fast response time by the clamp For the voltage electrode the pipette tip should be broken back to an O D of 3 5 um The current electrode pipette should be broken back to an O D of 7 9 um The resistances of these pipettes should be about 2 MQ and 1 MQ or less respectively 1 5 to 2mm re 5mm BREAK TIP TO DIAMETER OF 3 5 MICRON FOR V ELECTRODE 25mm 0 3mm OR 7 9 MICRON FOR ELECTRODE 0 6mm When installed the current electrode pipette should be shielded from the voltage electrode and that shield should be grounded to the circuit ground This can be accomplished by wrapping the current pipette with aluminum foil or by mounting a metal screen or plate between the two pipettes In either configuration the shield can be grounded by connecting it to the ground mini jack on the side of the bath probe When using the aluminum foil m
11. atus Company
12. b inspect the voltage electrode to see if there is a bubble in the pipette which will cause an open circuit Using the Vm OFFSET control adjust the VOLTAGE ELECTRODE potential to read 0 mV If the junction potential of the voltage electrode can not be adjusted to 0 mV the electrode holder may be at fault See Electrode Holders page 17 To test the resistance of the voltage electrode pipette depress the ELECTRODE TEST button This passes a 10 nA current across the voltage electrode The VOLTAGE ELECTRODE METER will display the resulting potential in mV The resistance of the electrode can be easily calculated by dividing the current into the potential The resulting answer will be expressed in Q For example if the electrode test indicates that a potential of 25 0 mV is produced by the 10 nA test current then pa ZMV 5 5x1050 25M0 I 10nA Warner Instruments A Harvard Apparatus Company Current electrode placement 7 8 9 Advance the current electrode until the tip is in the chamber bath solution Adjust Ve OFFSET for a zero reading on the CURRENT ELECTRODE METER This will establish a null reference allowing the resting potential to be directly read With the CLAMP SELECTOR switch in the off position the resistance of the CURRENT ELECTRODE pipette is tested in the same manner as the VOLTAGE ELECTRODE Pressing the Ve ELECTRODE TEST pushbutton will cause a 10 nA current to be passed across the CURRENT ELECTRODE
13. d Apparatus Company 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 A lit LED indicates the active status of this circuit 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 MO in parallel with 0 5 uF and 3 the compliance voltage of the clamp current Bath electrodes The BATH ELECTRODES control block contains the BATH PROBE connector the CURRENT METER the GAIN SELECT switches and the I MONITOR output BNC s The CURRENT METER reads the voltage Ve of d ev ion the current electrode when the CLAMP A BATH PROBE MODE SELECTOR switch is off see CLAMP section A lit LED indicates voltage readings in mV When in clamp mode CLAMP MODE SELECTOR switch set to slow or fast the CURRENT METER displays the current Im sensed by the bath electrode A lit LED indicates current readings in A Instrument gain is set by the two GAIN SELECT controls Gain is selected by a 7 position GAIN SELECTOR switch which ranges from 0 1 to 10 in 1 2 5 steps and a 3 position toggle switch which selects the gain multiplier x0 1 x1 0 and x10 LED s indicate the gain multiplier selection The combination of thes
14. d on the BATH ELECTRODES METER in units of uA Fast clamp speeds are 350 us when measured with the model cell as described above The GAIN control is a single turn potentiometer which varies the full bandwidth open loop gain from 0 to 2000 A high Dc GAIN 109 can be switched in with the DC GAIN toggle switch to provide a hard clamp when passing large currents from high expression oocytes Commands The COMMANDS control block contains the HOLD controls and COMMAND COMMANDS 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 Signal polarity or off is selected with the associated toggle switch COMMAND IN 10 input BNC Command signals from an external generator or computer connected to this input are attenuated by 10 Maximum input is 10 V Current electrode The CURRENT ELECTRODE block includes the Ve OFFSET ELECTRODE TEST and Ve BUZZ controls This section also contains the OVER VOLTAGE indicator and a DIN connector for the current electrode Warner Instruments A Harvard Apparatus Company Ve OFFSET With a range of 200 mV center zero this CURRENTE control is used to adjust the offset voltage of the Ve OFFSET current electrode Use this control to establish a zero reference before impaling the oocyte Once the oocyte has been pierced the resting
15. dditional DC gain of 106 may be employed for high conductance cells or leaky oocytes Two clamp speeds are available The Slow mode is used for screening oocytes or for applications not requiring rapid response times The Fast mode is used for accurate voltage clamping of fast whole cell currents Response time in the Fast mode is 350 us 10 90 rise time when applying a 100 mV step to a model cell Bath Clamp Headstage The current measuring range of the OC 725C bath clamp headstage is extended at both ends by a 3 position range multiplier This allows smaller currents to be amplified to usable levels while larger currents up to 1 mA can be recorded without saturation The unique design of the bath clamp eliminates the need for series resistance compensation It provides an accurate measurement of bath current by creating a virtual ground in the bath while simultaneously clamping the bath potential at zero 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 Vm voltage input Dual Oocyte Studies Studies involving two oocytes in a common bath requires two clamp amplifiers Traditional bath clamp headstages cannot provide effective clamping because they cannot separate the individual currents from the combined currents appearing in the bath The OC 725C solves this problem by the application o
16. e Vm Membrane potential may be recorded from the Vm x10 connector VOLTAGE ELECTRODE section Ve The voltage of the current electrode can be monitored from the Ve connector on the REAR PANEL The output will be the same as that reported on the CURRENT ELECTRODE METER showing the voltage across the current electrode when the CLAMP SELECTOR switch is in the off position Recall that the meter indicates the current Im when the camp SELECTOR switch is in either the slow or fast position Warner Instruments A Harvard Apparatus Company Im The current signal is available from the I MONITOR and I MONITOR FILTERED outputs BATH ELECTRODES section Gain telegraph Automatic monitoring of the Im gain can be achieved by connecting the rear panel GAIN TELE BNC to the appropriate input on your analog to digital converter Power The power cord should be connected to a properly grounded AC receptacle with the line voltage specified on the instrument nameplate REAR PANEL Resting position of the controls To begin set the instrument controls to the following positions Control Section Setting POWER off I MONITOR output BATH ELECTRODE 1 V uA GAIN SELECT BATH ELECTRODE xl HOLD COMMANDS 0 mV POLARITY toggle COMMANDS off MODE SELECT CLAMP off CLAMP DC GAIN toggle CLAMP out CLAMP GAIN CLAMP off fully CCW ALARM REAR PANEL off or on as desired Turn POWER on Usin
17. e DC and may need to be IR and UV filtered if you plan to use it during recording Minimally the recording chamber can be a stable surface on which the oocyte will not roll around A disposable petri dish with a piece of nylon mesh on the bottom has been successfully used for this purpose The dish can be stabilized by a holder or by some wax placed around its perimeter The diameter of the dish needs to be large enough to accommodate the oocyte and the two bath electrodes The walls of the recording chamber should be low enough to not interfere with electrode placement Perfusion of the chamber can be accomplished using a gravity fed system Perfusate from the dish can be evacuated using gentle vacuum Use as small an aperture as possible to avoid disturbing the surface of the perfusate in the dish 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 Finally a microelectrode puller is necessary for making appropriately sized voltage and current electrodes Usually the microinjection pipette puller can also be used to make microelectrodes You will need use of a microscope to break off the pipette tips Warner Instruments A Harvard Apparatus Company APPENDIX Specifications Equipment is intended to be operat
18. e controls allows gain to be set from 0 01 to 100 Current outputs are available from the 1 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 The CLAMP MODE SELECTOR switch selects for slow and fast clamp speeds or for off These choices are described below off In the off position the clamp amplifier is disconnected from the current electrode The voltage difference between the Warner Instruments A Harvard Apparatus Company current electrode and the bath electrode Ve mV is read on the METER in the BATH ELECTRODES control block This information is also available at the Ve x10 output BNC on the rear of the instrument slow The slow clamp mode is useful for screening of oocytes or where high clamp speeds are not required The slow clamp speed is approximately 0 5 ms when measured with the model membrane 1 MQ shunted with 0 47 uF In this mode measured currents are displayed on the BATH ELECTRODES METER in units of pA fast Most oocyte clamping is performed in the fast mode The clamp speed is limited by the resistance of the current electrode and the oocyte membrane capacitance Therefore the current electrode resistance must be kept as low as possible to obtain the fastest clamp speeds Currents are rea
19. ed in a controlled laboratory environment Voltage recording channel Vm Input Impedance Output Resistance Vm OFFSET Noise Electrode Test Negative Capacity Vm Meter Range full scale Bath electrode channel Im Ve OFFSET Noise Im Clamp clamp on Open loop clamp off MONITOR MONITOR FILTERED 4 pole Bessel Gain Telegraph Meter Ranges full scale Ve clamp off Im x0 1 range Im x1 O range Im x10 range Current electrode channel V Compliance Voltage Alarm Gain Variable DC Electrode Test Commands Hold internal External input attenuated by 10 Maximum external input Power requirements 100 130 or 220 240 VAC 50 60 Hz Dimensions Enclosure Voltage Headstage Mounting Handle Bath Headstage All noise measurements made with an 8 pole Bessel filter 5 x10 Q shunted by 3 pF 1000 200 mV at V probe input 50 uV RMS at 1 kHz 10 mV MQ 0 45 pF 199 9 mV 200 mV 5 5 nA RMS at 1 kHz x7 range 28 pA RMS at 1 kHz x7 range 0 01 100 V uA in 3 ranges 7 steps per range Same as above filtered at 1 kHz 0 2 2 6 VDC in 0 2 V steps 199 9 mV 199 9 pA 19 99 pA 1 999 pA 180 V 160 V 0 2000 AC DC 1x10 DC switch selected 10 mV MQ 198 mV in 2 ranges 1 V in 0 1 V command 10 V 9x42x25 cm HxW x D 1 25 x 5 cm dia x length with 1 8 m cable 4 8 mm x 6 3 cm dia x length 2 8 x 3 5 x 4 2 cm H x W x D with 1 8 m cable Warner Instruments A Harvard A
20. ed when the membrane resistance between the current electrode and the bath virtual ground goes to zero This will damage the oocyte For this reason we recommend that the user enable the audible overload alarm to provide a warning when the potential for such damage exists SPECIAL CIRCUMSTANCES High side current measuring In studies of single oocytes current is monitored by the bath clamp headstage Experiments involving two oocytes in a common bath such as gap junction studies requires monitoring currents from each oocyte This is done in the current output leg in series with and ahead of the current electrode Two disadvantages of monitoring the current in this manner exist a The noise level of this signal is higher However this is usually not a serious problem since currents are typically in the pA range b The voltage drop across the solution resistance from oocyte to bath ground becomes an error voltage since it is not subtracted out as when the bath clamp headstage is used 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 2 Remove the two screws at the rear of the top cover and it off 3 Locate dip switch S10 on the circuit board For normal operation S4 is on and all other
21. ee eese een setae tna tn aetas etos e esee esee se sete sete esse sas e tas ta setas sena 10 Initial instrument ECOUTER E P 11 Test DEOCeGUTFes eiecit A O NN 11 Offset Controls tussi RR ueniet uti iei i RE 11 Voltage electrode est nere lic aa RE 12 jn RR 12 Current electrode testa rte En vei t eres Wai E aN evan Pe RU PHAR Te Ces 12 DC UE RT 13 AC clamp iesti nicae AA RE E epe T e PR po e te ee 13 S1 DA B NJ M HE 15 Pipettes pM H 15 luongnocH 15 Bath probe anran EAA 16 Electrode placement and grounding sssesocecoesooessoecooecsocesocesocesocesoccssecsoesssessoecesecssecssecesessocesocssooses 16 Bath clamp electrode placement eese esee nan nn anno na anna na entente anna nc entes 16 Single oocyte setup with indirect ground esee eese eren ennt enne enne 17 OC 725C Manual Rev 050816 2 Single oocyte setup with direct ground esses eene eene tenente eene nne ener ens 17 D al ooctyesel Up iic eietbc iter ts eek bet Haee eoe EENE EL FR Ee ERR ERE HA Re EO Lea be HARI EAEE 17 Cable Ie 18 Resting position of the controls 4 ccce ee ee eee eese seen esta sette s eb aes ea eee pese co
22. erated as long as the button is depressed Current electrode test This test is performed using controls in the CURRENT ELECTRODE section Warner instruments A Harvard Apparatus Company With the model cell in place depress the Ve ELECRODE TEST pushbutton and observe a reading of 20 mV on the CURRENT ELECTRODE METER This corresponds to a 2 MQ reading at a calibrated response of 10 mV MQ With the CLAMP MODE switch set to off the BATH ELECTRODE METER monitors Ve voltage at the current electrode In this case Ve will be a measure of the voltage across Rm and the meter will indicate 10 mV 1 MQ DC clamp test This test is performed using controls in the CLAMP ELECTRODE section Place the CLAMP MODE switch in the fast position and adjust the Vm OFFSET control for a reading of 100 mV on the VOLTAGE ELECTRODE METER Turn the GAIN control on and slowly turn the control clockwise until the meter reading Vm decreases to zero The CURRENT ELECTRODE METER should read 0 10 uA Set the HOLD POTENTIAL COMMANDS section to 100 mV 50 mV on thumbwheel and MULTIPLIER toggle at x2 Select positive pos polarity The VOLTAGE ELECTRODE METER should read 100 mV and the CURRENT ELECTRODE METER should read 0 00 uA Switch to negative neg polarity The VOLTAGE ELECTRODE METER should read 100 mV and the CURRENT ELECTRODE METER should read 0 2 uA Return the GAIN control fully CCW and turn the CLAMP MODE switch to
23. ethod care must be taken to prevent the foil from touching the surface of the bath solution at the bottom end of the pipette or the silver electrode wire at the top end 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 KCI solution in the pipette must be chlorided to reduce junction potentials see Chloriding Warner Instruments A Harvard Apparatus Company Procedure in Appendix The pipette should contain just enough KCl so that approximately 1 2 inch of the chlorided wire is submerged The pipette holder assembly is attached directly to the voltage headstage prior to mounting in a micropositioner Current Electrode The current recording electrode also uses a silver wire for coupling In an manner analogous to the voltage electrode the current electrode wire must be chlorided prior to assembly and use The pipette holder assembly is mounted in a micropositioner with the mounting rod supplied Bath probe The bath clamp is designed to maintain a virtual ground in the oocyte perfusate The bath probe should be positioned so that the silver electrode wires can be inserted into the recording chamber or into the agar bridge wells Sticky wax or tape is usually sufficient to secure the unit when positioned on a flat surface or alternatively the unit can be held in place on a separate stand The ba
24. f an internal switch permitting measurement of the current in series with the current electrode instead of in the bath Additionally an optional differential voltage headstage is available which subtracts the voltage drop across the series resistance in the bath Voltage Headstage Probe The voltage measuring headstage is a single ended high impedance probe Its small size convenient mounting rod and two meter cable make for easy attachment to a micropositioner Warner electrode holders having a 2 mm jack mount directly onto the headstage Voltage and Current Meters Independent meters provide simultaneous displays of membrane voltage Vm and membrane current Im To assure proper impalement of the current electrode the current meter displays membrane potential Ve from the current electrode before the clamp circuit is activated 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 Warner Instruments A Harvard Apparatus Company 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 Since our goal is to provide clarity
25. g the gain select A wider range of bath current Im measurements is now possible with the addition of the GAIN SELECT toggle switch located above the GAIN control The switch has 3 positions x0 1 x1 and x10 Resistance values shown below each LED indicate the bath clamp feedback resistor used for the current measurement The chart on the next page shows the effect of the range selection on the Im output Note also that range selection changes the sensitivity of the current meter Typically measured currents will fall in the x7 range The lower and higher ranges are intended to cover those applications where currents are beyond the x range Currents below 1 uA should be monitored in the x10 range Large currents above 100 pA require the x0 1 range Since there is overlap in the ranges the current being measured may be monitored in one of two ranges in which case the choice may be made on the basis of noise or clamp speed Warner Instruments A Harvard Apparatus Company Gain select Headstage Im output range Im max output Maximum meter resistor V uA uA reading x0 1 10 kQ 0 01 1 0 10 1000 199 9 uA x1 0 100 ko 0 1 10 1 100 19 99 uA x10 1 MQ 1 0 100 0 1 10 1 999 pA Other gain range selection considerations The intrinsic noise of the current measuring circuit is a function of the bath clamp feedback resistor with a larger resistor offering lower noise and greater signal resolution For comparisons
26. in Figure A This method uses the bath clamp headstage to establish the bath ground and is preferred for two reasons 1 current readings with the bath clamp will have the lowest noise level and 2 properly placed bath clamp electrodes will negate the need for series resistance compensation CURRENT ELECTRODE 7 CURRENT ELECTRODE 4 HOLDER AND CABLE e HOLDERANDCABLE Vm HEADSTAGE L vor Vm HEADSTAGE Ya SHIELD CURRENT ELECTRODE J CURRENT ELECTRODE CLAMP CURRENT PATH CLAMP CURRENT PATH E BATH RESISTANCE BATH REFERENCE ELECTRODE BATH Figure A CLAMP HEADSTAGE Figure B 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 of the current output leg This method also requires the use of the optional DIFFERENTIAL VOLTAGE HEADSTAGE Two disadvantages are 1 The noise levels of the current signal measured in the high side is approximately double that obtained with a bath clamp and 2 high levels of clamp current could produce a substantial voltage drop across the solution series resistance Dual ooctye set up Dual oocyte setup is accomplished using two clamps as illustrated in Figure C Both clamps must be configured
27. ly 5 turns 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 This can be verified by rotating the control first fully clockwise and then fully counterclockwise while observing the VOLTAGE ELECTRODE METER The displayed readings will indicate off scale at the extremes of the control s manipulation since the meter is only capable of displaying 199 9 mV Vm x10 output BNC VOLTAGE ELECTRODE section This output can be monitored using an oscilloscope The reported voltage will swing between 2 V as the Vm OFFSET control is Warner Instruments A Harvard Apparatus Company manipulated throughout its full range Set the Vm OFFSET to 0 0 reading on the meter and verify that the Vm x10 reading on the scope also reads 0 V Ve OFFSET control CURRENT ELECTRODE section The Ve OFFSET control is tested in the same manner as the Vm OFFSET control This control is adjustable when the CLAMP MODE switch is set to off The voltage of the Ve OFFSET is read from the BATH ELECTRODE METER when the CLAMP MODE switch is set to off Ve x10 output BNC This BNC is located on the rear panel of the instrument and reports the setting of the Ve OFFSET control when the
28. mers Harvard Apparatus is committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for those Harvard Apparatus products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of Harvard Apparatus products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive Harvard Apparatus will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as they apply Special Collection Disposal Required Do Not Dispose Product with Municipal Waste Warner Instruments A Harvard Appar
29. n Finally hydraulically driven micro manipulators will also reduce membrane damage while the electrodes are in the cell Repeated recordings Most recording sessions will involve repeating the above steps several times with many different cells Unless there is a concern about contamination of the bath solution by something carried over from previous experiments the pipettes can also be used repeatedly They should be free of debris and should have approximately the same resistance as they had in the previous recording A significantly higher resistance could indicate that the pipette is partially plugged with cellular debris Make the following control settings before the next recording is carried out Control Section Setting POLARITY COMMAND off MODE SELECT CLAMP off GAIN CLAMP 0 fully CCW Electrophysiology If you are well versed in setting up electrophysiological equipment you can safely skip over the rest of this section If however this is your initiation into electrophysiology as it may well be for some of you molecular biologists then you may find the following recommendations helpful While the whole cell electrophysiological configuration is more forgiving than the patch clamp it is still important to minimize mechanical motion The platform for your experimental setup therefore should be mechanically well isolated This will reduce leakage around the electrodes making the clamp more effec
30. nclosure and connected to earth through the power line cord A shorting link allows for interconnection of the two grounds In most experimental setups separating the grounds will result in minimizing 50 60 Hz signal interference from ground loops However trial and error will determine the best results Warner Instruments A Harvard Apparatus Company Additional components Voltage recording headstages 7250V PROBE Standard Version The voltage probe is an active headstage housed ina 1 25 x 5 cm cylinder dia x length The probe body is nickel plated and epoxy sealed for corrosion resistance Warner microelectrode holders with 2 mm jacks mate directly to the input pin on the probe body A mounting block and handle are supplied and facilitate attachment of the probe to a micromanipulator The handle can be mounted either axially or perpendicular to the probe body 7255DI DIFFERENTIAL PROBE Optional This voltage probe is designed for applications where two oocytes share a common bath or where the voltage drop across the solution resistance is to be measured and subtracted from Vm The headstage housing is approximately 2 cm longer than that of the 7250V PROBE and has two additional inputs CIRCUIT GROUND and V DIFFERENTIAL When the two inputs are shorted the probe functions exactly the same as the standard single ended 7250 PROBE Bath headstage The BATH PROBE is housed in a 2 8 x 3 5 x 4 2 cm aluminum enclosure Inputs are
31. on If the buzz technique fails to cause electrode penetration further advance the voltage electrode until it pops through the membrane The potential across the membrane will now be displayed on the VOLTAGE ELECTRODE METER 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 10 kHz oscillation across the current electrode This disrupts the cell membrane and causes the tip of the electrode to impale the cell with no further movement of the micro manipulator Once again if Warner Instruments A Harvard Apparatus Company the BUZZ technique fails to cause penetration further advance the current electrode until it pops through the membrane Clamping the cell 14 Activate the clamp by switching the CLAMP MODE switch to either the slow or fast mode 15 Adjust CLAMP GAIN control clockwise as far as possible without illuminating the OVER VOLTAGE LED located in the CURRENT ELECTRODE section tm 16 The clamped membrane potential can now be observed over time or it can be manipulated by selecting the desired polarity and amplitude with the controls located in the COMMANDS section Alternatively you can control the COMMAND voltage externally from a computer by leaving the POLARITY toggle switch in the off position and connecting the appropriate analog o
32. potential can be read from Ve x10 output BNC or on the current meter BATH ELECTRODES section ELECTRODE TEST A voltage proportional to the resistance of the current electrode 10 mV MQ will be displayed on the meter by depressing the ELECTRODE TEST push button when the CLAMP MODE SELECTOR switch is in the off position Ve BUZZ The Vm BUZZ pushbutton facilitates penetration of the voltage electrode by producing a 10 kHz square wave at the pipette tip OVER VOLTAGE LED If the voltage at the current electrode exceeds 160 V the OVER VOLTAGE lamp will light An alarm will also sound when the rear panel ALARM switch is in the on position 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 ca TELEGRAPH output BNCs the ALARM switch and instrument GROUNDS Ow The Ve x10 output BNC monitors the voltage of the current electrode x10 when the CLAMP MODE SELECTOR switch is off The GAIN TELEGRAPH output BNC provides a DC voltage indicating the gain setting of the instrument The output varies from 0 2 to 2 6 volts in 200 mV steps as shown in the appendix ALARM switch Activates or deactivates the over VOLTAGE current electrode audible alarm GROUNDS Both CIRCUIT and CHASSIS grounding posts are located on the rear panel CHASSIS is common with the instrument e
33. pparatus Company Gain telegraph outputs Im Output V uA Gain Telegraph 0 01 0 2V 0 02 0 4 V 0 05 0 6 V 0 1 0 8 V 0 2 1 0V 0 5 1 2V 1 1 4V 2 1 6V 5 1 8V 10 2 0 V 20 2 2V 50 24V 100 2 6 V Gain select settings Gain select Headstage Im output range Im max output Maximum meter resistor V uA uA reading x0 1 10kQ 0 01 1 0 10 1000 199 9 uA x1 0 100 kQ 0 1 10 1 100 19 99 uA x10 1MQ 1 0 100 0 1 10 1 999 uA Noise from bath clamp feedback resistor Noise Feedback Resistor Shorted Input Standard Model Cell Modified Model Cell 0 5 pF 0 22 uF 10 kQ 75 pA 6 0 nA 44 nA 100 KQ 28 pA 5 5 nA 44 nA 1MO 22 pA 5 0nA 4 0 nA Warner Instruments A Harvard Apparatus Company 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 and Sigworth F J 1991 Multiple gating modes of the ul sodium channel Neuron 7 775 785 Warner Instruments A Harvard Apparatus Company Certifications Declaration of Conformity CE MARKING EMC Application of Council Directive 89 336 EEC Standards To Which Conformity EN55022 Class A Is Declared EN61000 3 2 EN61000 3 3 EN50082 1 199
34. sets eR a seen 00000000 ease etas 19 Using the DII V iSo csatos 19 Other gain range selection considerations eee esee eere e eee ee ee enne seta sette seta aset ease tates etas esten aee 20 A PROCEDURE FOR RECORDING FROM OOCYTES eerie sees eese teet ee eo setae ttes setas e testae en aes 21 Initial electrode placement 0 2000 000022000000000000000200082000000000000000000050100000000000000800008000000000 000000000 21 Voltage electrode placement 2 aaa Era casae va Te sa Va Te sa soe Eua b eR Te a eo There ee 21 Current electrode placement 0 02000s000ss00000000000000s200000200000000000000s200000000000000000008000080000000000 0000000000000 22 Impaling the cell e M 22 ei noPAltacjee E eesse 23 Clamping high conductance cells 4 eee eee eee eee e eene eene eene etate tense ta seta cono conocconaconaconncconeconos 23 Uniclamiping the Cell nC 23 O a AAA ts tris co sesvecnooi esov to cece ti vess sri nos svesno irosit Seske 23 SPECIAL CIRCUMSTANCES asisisscsdsccseccavsssccccscsseccsascosceecconesctestssetsecesssescsesbeustecsoscveccoseteecesvectsadsseteceses 24 High side current measuring eue cose toco oae to eroe nino east en tese nt Vosa voca Uoc ee real osa eo oerip rares e 24 ConfigurattQms z odii e ede Hle Rago AA etd t oe Ra oe 24 Optional voltage headstage corssonssonssons
35. ssnsesnssnnnennussnnssnnssnnnsnnssnnnsnnnssonssonsssnnssnsesnnssnunsnnssnnssnnssnnssnnne 24 COMMENTS AND RECOMMENDATIONS eee eee esee eene en ette sone en otto ste ense tust te tee ease tastes eese taste soa 26 MENTES O 26 Repeated recordings sesssisccccsssziscensccsosseessescensccseseesessecenscessscoocedvesscessesosgsctenssessoonascedsescedesenbssestersnessens 26 El ctr ph siology 26 APPENDIX iiss E NO 28 Specifications Equipment is intended to be operated in a controlled laboratory environment 28 Gain telegraph Outputs eereo eee ie tno ron voro so tos ono essnee orase eae on eV unen tea nsee ee eE eue a apu ece apte eher 29 Gain select Settlligs esee roe NN 29 Noise from bath clamp feedback resistor 1 eee ee ee ee eee eere eee ee eee tosta esten aset tn sette setas seen 29 References poo 30 Certifications EC 31 SE TERRIER 31 Unique Features Additional Features The model OC 725C 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 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 The AC clamp gain is variable up to 2000 An a
36. th probe electrodes should also be chlorided before use as described above Electrode placement and grounding Three drawings shown on pages 19 and 20 have been included to illustrate the various ways a bath circuit can be configured Most applications involve only a single oocyte and Figures A and B illustrate these setups Figure C shows a setup for recording from 2 oocytes in a common bath with the use of dual clamps 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 The Iout electrode or the agar bridge associated with it on the other hand can be placed at a greater distance from the oocyte and should be on the same side as the current electrode It is recommended that the user not directly expose the electrode wires to the perfusate if the recording session is to last for more than a few minutes Instead agar bridges should be employed to provide a circuit between these electrodes and the bath This protects the cell membrane from the potential adverse effects of the silver wire Warner instruments A Harvard Apparatus Company Single oocyte setup with indirect ground Single oocyte studies are best accomplished with the setup shown
37. tive and reducing noise in your recordings The latter is especially important when recording responses of certain ligand gated channels where membrane potential changes may only be a few mV 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 Warner Instruments A Harvard Apparatus Company 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 Then only one wire is run from the setup to the instrument ground You will need to mount the voltage recording electrode headstage and the current injecting electrode on micro manipulators They need not be hydraulically driven but such drives will minimize the damage to the cell during and after penetration and will make for better seals around the electrodes Another advantage gained by reducing membrane damage by electrodes is to enhance the possibility of making subsequent recordings from the same cell These suggestions are also important for minimizing mechanical noise in the recorded data You will need a dissecting scope for viewing the placement of the electrodes Anything more powerful than 40x will just get in the way The light source for your scope should b
38. to read current from the high side as described in the section titled HIGH SIDE CURRENT MEASURING see page 26 and each clamp must be equipped with the optional DIFFERENTIAL VOLTAGE HEADSTAGE Warner Instruments A Harvard Apparatus Company pe CLAMP 1 poe e A CLAMP 2 A CURRENT ELECTRODE HOLDER AND CABLE CURRENT ELECTRODE HOLDER AND CABLE Vm HEADSTAGE CURRENT Vm ELECTRODE 1 N j q ELECTRODE 1 ELECTRODE 2 CLAMP CURRENT PATH BATH RESISTANCE NI A NN V DIFF ELECTRODE 2 A a V DIFF ELECTRODE 1 BATH REFERENCE ELECTRODE Figure C 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 High voltage current electrode The holder should be mounted on a micro manipulator and the cable connected to the I ELECTRODE socket CURRENT ELECTRODE section Command potential If a computer or external generator is used for controlling the clamp command potential its signal should be connected to the front panel COMMAND IN 10 input COMMANDS section External monitoring To monitor the microelectrodes potentials on an oscilloscope computer or a chart recorder the following connections should be mad
39. tors for GAIN TELEGRAPH OUTPUT and V x10 output an ALARM on off switch and binding posts for CIRCUIT and CHASSIS GROUND Voltage electrode The VOLTAGE ELECTRODE control block contains VOLTAGE ELECTRODE Vm Vm mv the VOLTAGE PROBE CONNECTOR the VOLTAGE HO METER the Vm OFFSET control the ELECTRODE E G x TEST and BUZZ pushbuttons and the Vm OUTPUT E c BNC Controls for NEGATIVE CAPACITY p vm COMPENSATION are also located in this block rose VMOFFSET gc Vm X10 The VOLTAGE METER reports the membrane AS e voltage Vm with a full scale range of gt 199 9 mV The VOLTAGE PROBE CONNECTOR is a 7 pin DIN connector for attachment of the voltage probe to the instrument The Vm OFFSET control is a 10 turn potentiometer providing up to 200 mV at the VOLTAGE PROBE input for offset of membrane junction potentials The Vm ELECTRODE TEST control is used to determine the internal resistance of the voltage probe When the pushbutton is depressed a constant 10 nA current is passed through the voltage electrode producing a voltage drop of 10 mV MQ of probe resistance The measured potential is displayed by the meter or reported at the Vm x10 output BNC at 100 mV MQ The Vm BUZZ push button facilitates penetration of the voltage electrode by producing a 10 kHz square wave at the pipette tip The Vm x10 ourPUT BNC reports the membrane voltage in mV multiplied by 10 Warner Instruments A Harvar
40. two 1 mm pin jacks labeled I SENSE and I our The case is electrically grounded and a pin jack is located on the side for connecting to shields A plastic plate with two screw mounting slots is attached to the probe base The BATH PROBE connects to the control unit with a 6 pin connector Current electrode cable A two meter shielded cable is supplied with a 2 mm pin jack on one end to mate with the an electrode holder and a 3 pin connector on the other end to mate with the instrument The electrode holder should have a handle for mounting to a micromanipulator An example is shown to the right Warner Instruments A Harvard Apparatus Company Model cell The model cell supplied with the OC 725C is useful as a training aid and as a calibration and test device It has connections for the voltage and current probes and to the bath clamp allowing all aspects of the amplifier s function to be tested 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 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 USING THE MODEL MEMBRANE The model cell supplied with
41. utput from your computer DAC to the COMMAND IN 10 BNC connector See Cable Connections page 20 17 Depending on the amplitude of the response you wish to record you may adjust the instrument GAIN to a higher or lower position The CURRENT ELECTRODE METER should now be displaying the current in uA that is delivered to hold the cell at the designated command potential Clamping high conductance cells 18 DC GAIN mode CLAMP section may be required to clamp high conductance low resistance cells This condition will be evidenced by the inability of the instrument to maintain a DC holding potential to within 1 or better of the set value and the maximum instrument gain is not sufficient to provide a hard clamp DC GAIN mode provides an additional DC gain greater than 10 while the AC gain remains at 2000 maximum for stability 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 O Removing the electrodes 20 It is very important that the CLAMP GAIN be returned to the off position fully counter clockwise to click off as described above and the CLAMP MODE selector switch be placed in the off position before removing the current electrode from the cell Warner Instruments A Harvard Apparatus Company Failure to perform the above steps will overload the feedback amplifier due to the large current generat
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