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EC-825A User`s Manual

1. 21 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Place the HEADSTAGE VOLTAGE MEASURING ELECTRODES into their respective chambers Turn the HEADSTAGE SELECTOR switch to on and select the desired measuring mode see below Recording modes The recording mode to be used voltage clamp or current clamp depends upon the experimental design Advantages and disadvantages of different recording modes are listed below Amplify In this mode the current passing amplifier is disconnected from the current passing electrodes HEADSTAGE I1 and I2 INPUTS This mode is used to measure any asymmetry in the voltage sensing electrodes Since the current passing amplifier is disconnected an external voltage or current applied to the EXT CMD input BNC will not be passed across the epithelium Voltage clamp In this mode the transepithelial voltage is set to a specified value and the amplifier passes a transepithelial current to maintain that voltage The magnitude and polarity of the clamp voltage is set within the COMMAND control block Voltage clamp mode can be used for example to determine which ions are actively transported by the epithelium using radio isotopic flux measurements or pharmacological ion transport blockers Current clamp In this mode the transepithelial current is typically clamped to zero i e there is no net transepithelial current flow the condition the tissue is exposed to in vivo The
2. Ranges Internal Clamp Potentiometer 100 mV with 10 turn control External Command 1 V External Command factor 1 mV 10 mV applied Current Clamp Ranges Internal Clamp Potentiometer 1 mA with 10 turn control External Command 10 mA External Command Factor 1 pA 10 mV applied Speed 10 us measured with model membrane Compliance 50 V WARNER INSTRUMENTS A Harvard Apparatus Company Resistance Fluid resistance Compensation range 0 100 O standard 0 1 KQ optional Membrane Resistance Measurement made with a 2 Hz bipolar constant current square wave Ranges 0 2 KQ injected current 10 pA 0 200 KQ injected current 1 pA Panel Meter 3 5 digit LED Voltage Range 199 mV maximum Current Range 1999 pA maximum Timers A amp B Range 10 ms to 1000 seconds set with 2 digit resolution and 4 ranges each channel Power Requirements 100 130 VAC or 220 240 VAC 50 60 Hz Physical Dimensions EC 825A 9 x 42x 31 cm HxW x D Headstage 7 7 x 7 7 x bcm HxWxD Shipping Weight 10 1 kg Warrantee Three years parts and labor 37 EC 825A Rev 120924 1 38 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 1992 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
3. Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Equipment Description Instrument Amplifier Equipment Class ITE Class A Model Numbers EC 825A I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Connecticut USA Signature Tiled Full Name Ralph Abate Position Director of Operations 38 EC 825A Rev 120924 1 39 Declaration of Conformity CE MARKING LVD Application of Council Directive 73 23 EEC Standards To Which Conformity Is EN61010 1 2001 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 EC 825A I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Connecticut USA Signature d Full Name Ralph Abate Position Director of Operations 39 EC 825A Rev 120924 1 40 WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers 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 Dire
4. en e Mee en ie OE ete it 11 Additional components and comments e eee eee ee eee ee ee eee seen aee ea sette etna esten aset ense esee sense eaa 12 Connecting to line power ground eeeseeeseeeeeeeee seen eene enhn enne tenet nene VENEET EEk ri nenne 12 Heddstdge te t Eee eee o age t e peg isle obese bla ede apte eia Led ee one reais 12 Connecting the headstages eese sees ense ene nennen en tene enne sten ss ette seen testen sienne eene ne en 13 SETUP AND INITIAL TEST 4 eeeeee cesse eee en senses enata stessa tns ta sens sos e tastes oa sees o aros sese sesso setas tns ena 14 Imi E HY 14 Chamber p HE 14 Electrodes a cte ete RR E MEO Ree e eI PAR e eere e ee e Ue e teo es 15 Ele CIEORQS s o ete Crete eA ete p ettet b e Or toa Ate P e ORR alee eR Tes 17 nid ure c 19 I tial controlsettilgs cie e e RN Eee RE ERREUR EXE Ye SER 19 Monitoring model membrane voltage eese eee esee eene enne entente tenens en nennen 19 Checking zero voltage clamp eese EEEE enne tenete sienten teens nein ne nien 19 Check voltage clamp command esses esee eene en tene enne tens sene niente tenete entente 19 Checking clamp current on panel meter eese eee nene 20 Fluid Resistance compensation eese eese eene nene entes en testen E E EEEE aa 20 EC 825A Rev 120924 1 OPERATION tS 21 Connecting the EC 8254 sis
5. of an epithlial membrane eese 30 Measuring epithelial surface area esses nren nennen nennen nnne 31 Further considerations seh e e eie t teer teli EEA Ce Dah ee deve 3l Liddlbqee p EHE 33 References and recommended reading eee eee eee ee eee ee eren ee ee eese to testen aestas etate seta ase eaas 33 Chloriding silver WIEGS uero ee ero aereo cepa eta egeta ee n Puer ra eee ee eun ed aea eiee ee o Pee ue ae eva een eae on ve Seen SESS rss 33 Adjusting the fluid resistance measurement range ecce eerte etes eee sete ette setae ta seta setas etas tassa 34 Connecting the EC 825A to Acquire amp Analyze eee e eere ee eee ee enne etn eese seta aset ense etes tasse tense 34 Hardwake connections 5 e de te Cec ia silted a acd oe Tet ente aeree e veu e orb ae 34 NYeL AIEO DITIITE ERE TEE 35 Specifications E ee eoe toin tbi rhe Feo Ce on Ene esu S ase e Pee Eee EN ENS HYPER EY RERE S E E E EEE 37 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company The EC 825A Epithelial Voltage Clamp from Warner Instruments is a two channel device providing accurate measurement of transepithelial voltage short circuit current and membrane resistance Important features include fluid resistance compensation membrane resistance readout high voltage compliance and small watertight headstages Operating modes include voltage clamp current clamp vo
6. or CHANNEL 2 Connect the DB15 to the associated port on the rear of the EC 825A and each DB9 to its respective CLAMP input on the MOD08 A Software configuration 4 5 Install and launch Acquire amp Analyze Following the instructions on pages 16 17 of the Acquire amp Analyze manual open a new experiment File New Experiment and name it This will bring up the Experiment Properties dialog box Make sure the number and location of Active Tissues selected corresponds to the number and location of clamp inputs on the MODO8 A e g 1 and 2 for the EC 8254 Next open the Instrument Settings Dialog Box Setup Instrument Settings and verify that the Voltage Current and Signal Gains are all set to 10 Click on the Advanced Settings button Set the Instrument Type to Custom Set the OPEN CLOSED CURRENT and VOLTAGE CLAMP patterns to O 3 O and 48 respectively Also uncheck the Manual Reference checkbox Click on OK twice to exit to the main screen On the amplifier set the following switches to the specified settings Control Setting Offset off Command off Mode ext timer Clamp response slow Headstage s Test mode Vmem 10 11 12 In the software open the Pulse Setup Dialog Box Setup Pulse Setup In the Clamp Mode section select Voltage Clamp and hit Apply Verify that the amplifier is now in voltage clamp Verify that the meter reads voltage O mV and c
7. resistance Moreover each headstage is equipped with a built in model membrane allowing for a rapid and convenient instrument test See page 17 for test procedures Connecting the headstages The headstage connects to the instrument via an 8 pin DIN connector in the CLIPPING control block To reduce noise the headstage should be placed as close to the test site as possible 13 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company SETUP AND INITIAL TEST The purpose of this section is twofold First a description of the equipment needed to measure the basic electrical properties of an epithelium is provided Second a series of procedures are provided to test the performance of the instrument Equipment The equipment required for the study of any epithelium including tissue cultured epithelia can be divided into three components The first component is a pair of hemi chambers typically termed Ussing chambers in which the epithelium acts as a partition between the two chambers Thus the epithelium separates two compartments the mucosal or luminal compartment and the serosal or blood side compartment The second component contains the electrodes for sensing the epithelial voltage and for passing current and the third component is comprised of the instrumentation necessary for measuring both voltage and current Chambers Irrespective of the detailed design useful chambers must have several common feature
8. resistance or conductance Experience has shown that a plot of Vi vs Rt is more sensitive to changes in either Rj or E than a plot of G vs Isc The reason for this is that whereas Is is a measure of only the cellular pathway it is not affected by the junctional resistance V is a function of both the cellular pathway and the junctional resistance and is thus more sensitive to a change in either parameter This is illustrated on the next page Figure 3 in which the experimental conditions were such that both R and E are changing Note that although the G vs Isc plot is linear the plot of Vi vs Rt is non linear Three approaches have been used to alter R in a controlled manner These are increasing the cell membrane 27 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company conductance to a given ion using second messenger systems decreasing the conductance to a given ion using pharmacological blockers and lastly artificially increasing the membrane conductance using pore forming agents such as gramicidin D nystatin or amphotericin B When using pore forming agents one must use a mucosa solution which mimics the cell interior i e the potassium content must be high while the calcium sodium and chloride content must be low Typically one performs an equimolar replacement of sodium with potassium and chloride with a large monovalent anion such as gluconate Such a solution exchange has two advantages First sin
9. s labeled A OUT j B OUT SYNC OUT and TIMER OUT Input BNC s include EXT TIMER IN for channel 1 and channel 2 e A OUT B OUT These BNC s report the active status of the TIMER A and TIMER B control blocks respectively A TTL high signal indicates on status and a TTL low level indicates off status for the respective TIMER e SYNC OUT This BNC is useful for synchronizing an oscilloscope with the EC 825A while monitoring output signals Connects to the oscilloscope trigger input e TIMER OUT This BNC reports the functional state of TIMER A or TIMER B depending on which timer is currently active For TIMER A TTL high indicates Zero V Clamp mode and TTL low indicates Zero C Clamp mode For TIMER B TTL high indicates V Clamp mode and TTL low indicates C Clamp mode e EXT TIMER IN Activated by selecting Ext Timer in the MODE control block Separate inputs are provided for CHANNEL 1 and CHANNEL 2 A TTL signal at this BNC switches the associated channel between voltage clamp TTL low and current clamp TTL high modes 10 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company I O interface This 15 pin D connector allows the EC 825A to be connected for computer operation D Pin designations are 1 EXT CMD IN channel 1 GATE IN channel 1 MEMBRANE RES kQ channel 1 V MONITOR Vm X 10 channel 1 I MONITOR 10 mV n4A channel 1 circuit ground channel 1 E
10. this voltage change AV is stored together with the corresponding time t where t O is the time at which the current was applied AV is then fit to a sum of exponentials using standard non linear curve fitting routines The number of exponentials used is limited by the equivalent circuit In general analysis will yield two voltage terms which are converted to resistances by dividing the voltage by the amplitude of the current pulse and two time constants t each of which is the product of a resistor and capacitor The relationship between the best fit values and the actual epithelial resistors and capacitors depend upon the equivalent circuit model used to represent the epithelium This is a problem of the non uniqueness of these equivalent circuits Further considerations Let us now consider the basic epithelial equivalent circuit model In Figure 4 we show two circuits The general form of the equations describing the voltage response of these circuit to a square current pulse are identical The relationship between the resistors and capacitors in these circuits is described by the following equations see Lewis and Demoura 1984 CC CC C C C C R R _ R R R C R C R C RC 1 1 R R RK R RC RC R RC RjR4C 31 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company 1 R R Ry R C R C RRC Ry Cp l Note that a five parameter model for simplicity we ignor
11. where A is the area of the epithelium and the units of resistance are in ohms cm Since there is a finite distance between the voltage measuring electrodes and the epithelium the calculated resistance Rmeas is the sum of the transepithelial resistance Ri 24 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company and the series resistance of the solution Rs i e the resistance of the bathing solution between the tissue and each of the voltage measuring electrodes This series resistance must be subtracted from Rmeas To determine the actual transepithelial resistance Rt Rmeas Rs the EC 825A can automatically subtract the series resistance once it has been measured from Rmeas Series resistance is usually determined by measuring the resistance of the chambers in the absence of an epithelium Since solutions of different ionic composition have different resistivities Rs must be uniquely determined for each solution used Although for high resistance epithelia series resistance is only a minor correction e g 1 296 of Rmeas for low resistance epithelia it can be 50 or greater of Rmeas An alternative approach for measuring R is offered on page 26 Short circuit current The measurement of the short circuit current Isc is deceptively simple Isc is simply defined as the current that must be passed across the epithelium to reduce the transepithelial voltage to zero It is the current that short circuits the tiss
12. 1 WARNER INSTRUMENTS A Harvard Apparatus Company vii Multi functionality Ideally the chamber should be multi functional Thus in addition to measuring transepithelial electrical properties it should also be constructed to allow for the use of micro or ion selective electrodes The former electrodes are essential for determining the individual membrane resistances while the latter are required to determine membrane ionic permeabilities viii Additional features might include the ability to measure cell volume and use intracellular fluorescent dyes Two chamber designs currently in use for the study of epithelial transport are shown below Solution outlet m Agar bridge m Solution inlet m Current electrode m d Agar bridge s Solution outlet s Solution inlet s Plexiglas insert aerator aerator Current electrode s outflow Teflon dish Silicone spacers Glass coverslip gt steel securing ring Figure 1 Two basic chamber designs for studying epithelial transport Left this is the original design by Ussing This chamber circulates solution across the surface of the epithelium using a gas lift system which also aerates the ringers and can be temperature controlled A disadvantage of this system is that it is closed and thus does not meet criteria vi and vii In addition the original design caused significant edge damage se
13. 5A within this control block LED s are provided to indicate overload conditions within the clamp amplifier or input amplifier circuitry Left and right LED s indicate positive high or negative low clipping states EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Meter The METER section is comprised of a 3 5 digit LCD and 2 position METER MODE toggle switch The METER MODE toggle switch selects between current and voltage readings Full scale is 1 999 uA and 199 mV for current and voltage positions respectively Current or voltage readings can be displayed for any selection within the MODE control block with the exception of MEMBRANE RES In MEMBRANE RES mode the meter displays the membrane resistance in either O or kO as selected by the associated MEMBRANE RES scale toggle found in the MODE control block Command Applied current or voltage amplitudes are set using this control Polarity of the applied signal is set by the associated toggle switch Placing the toggle switch into the center position turns this control block off This control is inactivated by a TTL high setting at the GATEIN BNC In voltage mode V CLAMP this control sets the holding potential from zero to 100 mV with the polarity set by the toggle switch In current mode C CLAMP this control sets the holding current from zero to 1 mA with the polarity set by the toggle switch Mode The MODE block is
14. EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Warner Instruments Dual Channel Epithelial Voltage Clamp Model EC 825A Warner Instruments 1125 Dixwell Avenue Hamden CT 06514 800 599 4203 203 776 0664 203 776 1278 fax EC 825A Rev 120924 1 Table of Contents NOMENCLATURE seners esre opresa aa eroas PoE SES Ep S aIo Sa So esiosa S 5 CONTROL PRIUS TOL FE COOL P sais 6 Channel 1 and 2 control blocks iiiter toe e abis o noie peo PFeH Rua eru o en E eu U ee tacussoususuadscssseuboaseussecsessuase 6 OLSEN eee eo o dte Liao eve ofer tru cde E le eee oA t t o de eee 6 CIDDIAu gi RD SERRA BERI EE edet BEARER 6 2E Z Command iure nee ERR UR anos Soa EET RE M EE Petts 7 MOE PEE AE as ees RE OG Re eR RR RI RR a RPM EE 7 Fluid ADAE UE amp Clamp Response 2c eae Bos rer e tates e ese ae RE E Ano dete tte acero eee E 8 Timer control bloeks i eoiscistre topi Pret reo PER t vb PXrE sro X ri S PORE UE REPAEVES FUE ETUR e PEDE S s VETRPEE V EP SEE ER SERIE 9 Timer Cr 9 ALTAE 9 R ar pan l description eee eere eoo rene o Scio eost ette an erro ee Son FR Euo oree S S esise eee pea epe Qva rens ENSS 9 Grounds line power connector and fuse eese eene nennen ener enne nen 9 Tumerisectlon use se eei eS seria tex i toit eed neve e adeo te E to edet pt p eve ae ed Eee vn 10 I O inteVjQGO 325a d dote Ai e ated e ted kv m edt uc Le etie 11 Channel BNO sae e ee Rae HORROR e
15. PUT BNC instructs the EXT TIMER IN BNC to select between the values set on the front panel for voltage or current clamp modes A TTL high signal at the GATE INPUT BNC selects instructs the EXT TIMER IN BNC to select for zero current clamp for current clamp mode or zero voltage clamp for voltage clamp mode Timer This setting activates the timer functions and allows the operator to either voltage or current clamp the epithelium at zero for a programmed time set by the THUMBWHEEL switch and RANGE selector of TIMER A and then either current or voltage clamp the epithelium at a preset current or voltage using the clamp command settings for a programmed time interval set by THUMBWHEEL switch and RANGE selector on TIMER B Four different settings are available in TIMER mode zero voltage clamp switched to a selected voltage clamp value zero voltage clamp switched to a selected current clamp value zero current clamp switched to a selected current clamp value zero current clamp switched to a selected voltage clamp value The magnitude and polarity of the selected voltage or current clamp value is set by the calibrated dial and polarity switch of the COMMAND section Warning lights If the voltage between the HEADSTAGE V1 and V2 INPUTS exceeds 1 2 V there will be an audio warning and visual warning that the input amplifier is saturating Similarly a saturation of the clamp amplifier has both audio and visual warnings Fluid resistance The resista
16. S A and deMoura J L C 1984 Journal of Membrane Biology 82 123 136 e Ussing H H and Zerahn K 1951 Acta Physiologica Scandinavica 23 111 127 e Wills N K and Clausen C 1987 Journal of Membrane Biology 95 21 35 e Methods in Enzymology Volume 192 Biomembranes Part W Cellular and Subcellular Transport Epithelial Cells 1990 An excellent book that gives detailed methods used to study a variety of epithelia Includes a discussion on such topics as edge damage and solution resistance e Methods in Enzymology Volume 171 Biomembranes Part R Transport theory Cells and Model Membranes 1989 Similar to the above book Includes good chapters on solution resistance edge damage artifacts epithelial impedance liquid junction potential etc Chloriding silver wires Before using Ag wires as current electrodes they must be chlorided New previously unused wire should first be cleaned with ETOH before continuing while previously chlorided wire should have the old chloride coating removed Two methods are commonly used to chloride Ag wire soaking a clean wire in household bleach or electroplating a clean wire using a voltage source Both methods are described below A Soaking in bleach Simply immerse the wire in full strength common household bleach Clorox for 15 to 30 minutes until a purple gray color is observed Rinse and use B Electroplating Electroplating a silver wire with chloride is achieved by making the wire positi
17. XT TIMER IN channel 1 TIMER ground iO 0 d aD a BY WY ND EXT TIMER IN channel 2 10 circuit ground channel 2 11 EXT CMD IN channel 2 12 GATE IN channel 2 13 MEMBRANE RES kQ channel 2 14 V MONITOR Vm X 10 channel 2 15 IMONITOR 10 mV nA channel 2 Channel BNC s This block contains non TIMER I O BNC s for each channel of the EC 825A CHANNEL 1 10 mVIuA Each row of BNC s is dedicated to a single channel Output BNC s report the membrane resistance the membrane potential and the membrane current Input BNC s allow the application of external commands and inactivation of the COMMAND control block on the front panel 11 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company K OHMS This is an output BNC reporting the membrane resistance when Membrane Res is selected in the front panel MODE block Output units are 1 mV Q or 10 mV kO depending on if O or kO respectively is selected on the associated MEMBRANE RES toggle switch V MONITOR Vm x 10 This is an output BNC reporting the membrane voltage in units of 10 mV mV This BNC is always active I MONITOR 10 mV uA This is an output BNC reporting the membrane current in units of 10 mV uA This BNC is always active EXT CMD This is an input BNC allowing external voltage or current clamp command input Applied current or voltage commands are summed with their respe
18. anner E R Se V _R V rearranging yields f E c 4 c and 26 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company rearranging yields 1 B R R 1 1 1 R R R c J substituting the above two equations yields LANES E R If a perturbation which changes only the cell resistance is performed a plot of the resulting paired values of Vi and R during this perturbation yields a linear double intercept in which both Rj and E can be determined see Figure 3 An important question is how does one know that the perturbation has altered only the cell resistance and not the junction resistance Rj or the cell voltage source Ec The best indicator will be that the plot is linear since a curvilinear plot suggests that the assumption of constant Rj and E has been violated A similar relationship can be derived if the experiments are performed under short circuit conditions The derivation is shown below Recall that G G G j and J EG or G ds f E Substituting gives Using this equation a plot of the transepithelial conductance G versus the measured short circuit current Isc will have an intercept equal to the junctional conductance the inverse of the junctional resistance and a slope equal to the inverse cell voltage i e 1 E This equation has the same assumption as the previous plotted equation i e the experimental perturbation only changes the cellular
19. ce there is low chloride in the bathing solution cell swelling due to KCl influx is minimized and secondly since the mucosal and cell ion concentrations are matched there will be no change in the apical membrane voltage when the pore forming agent is added i e E will not be affected as R is decreased Additionally since the ion concentrations are matched the value of E will be approximately equal to the value of the voltage source of the basolateral membrane 28 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Gy pS Figure 2 Plot of V versus R left panel and G versus lso right panel In this example the cell membrane resistance of the rabbit urinary bladder epithelium was decreased using the pore forming antibiotic gramicidin D Of importance is that the mucosal solution was designed to mimic the ionic composition of the cell interior and as a consequence increasing the apical membrane resistance will not alter the cell EMF This is confirmed since both plots are linear and yield near identical values for E and Rj Figure 3 A plot of V vs R left panel and Gi vs Isc right panel In this example the cell membrane resistance was decreased using gramicidin D In addition the mucosal solution was selected such that the cell EMF E changes during gramicidin action on the cell resistance This is illustrated by the fact that the plot of V vs R is non linear and indicates that one of the assumpt
20. comprised of a six position MODE SELECTOR switch and associated LED indicator lights LED s indicate which MODE function has been selected The meaning of available MODE options are shown below Available MODE options are V Clamp Selects voltage clamp mode Amplify With the exception of fluid resistance measurements the clamp amplifier is disconnected from the headstage resulting in no signal at the EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company headstage 11 OUTPUT This is a true amplify operation C Clamp Selects current clamp mode Membrane Res Selects membrane resistance measurement mode A 2 Hz constant current bipolar square wave is passed through the membrane 10 pA in the 0 2 kQ range and 1 pA in the 0 100 kO range and the membrane resistance is displayed on the panel meter which scales automatically with the range selected Resistance values are also reported to the K OHMS BNC on the rear panel The associated MEMBRANE RES scale toggle switch selects between display units of O or kO Ext Timer External timer mode A TTL signal applied to the EXT TIMER IN BNC located on rear panel will switch the amplifier between current clamp TTL high and voltage clamp TTL low modes Int Timer Internal timer mode The programmable internal timer switches between current clamp and voltage clamp modes Fluid Resistance This control is
21. ctive counterpart in the front panel COMMAND control block In voltage clamp mode the ratio of EXT CMD voltage to applied clamp voltage is 10 mV mV In current clamp mode the ratio of EXT CMD voltage to applied clamp current is 10 mV uA This input is inactive when TIMER A is on or when GATE INPUT is TTL high GATEINPUT This is an input BNC allowing control of command inputs and selection of zero current mode All clamp commands current voltage and external are deactivated when a TTL high signal applied The same TTL high signal selects zero current mode Additional components and comments Connecting to line power ground The EC 825A is supplied with a 3 conductor line cord One conductor provides a connection between the instrument housing and the earth ground Safe operation of this instrument will be assured provided that the power outlet is wired correctly and is connected to earth If the ground pin of the line cord is removed for any reason the instrument chassis must be connected to earth ground using a separate heavy gauge 14 or larger ground wire Headstage The EC 825A supports operation of two channels each with its own headstage Headstages have inputs for the 11 V1 v2 and I2 ELECTRODE cables and a three position switch selecting on off or test modes Furthermore test mode has a two position EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company toggle switch selecting for Vmem or fluid
22. ctives 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 40
23. e ii Right this chamber is designed to be used on an inverted microscope and meets all of the listed criteria including the ability to measure transepithelial electrical properties and monitor intracellular ionic activities using fluorescent dyes A disadvantage of this design is that solutions must be continuously flowed through the chamber and that this solution must be preheated Electrodes Electrodes are an essential component of any electrophysiological set up since they provide the low resistance interface between the Ringer s solution and the electronic equipment This section considers the electrodes used to sense the epithelial voltage and to 15 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company pass a transepithelial current Although it may seem trivial careful choice the electrodes used is important Some guidelines are listed below Choices One has three choices in voltage measuring electrodes These are silver silver chloride Ag AgCl electrodes calomel electrodes or agar bridges Which electrode set to use The answer to this question depends upon the epithelium to be studied as well as the composition of the solutions bathing the epithelium i Silver silver chloride Ag AgCl electrodes can only be used if the epithelium is not sensitive to trace levels of Ag ions e g toad urinary bladder epithelium is very sensitive to trace levels of Ag and if the Cl concentration activity in the solut
24. e the solution resistance R can be determined by 4 parameters To curve fit this five parameter model to the impedance data we must have an independent estimate of one parameter For example Clausen et al 1979 measured the ratio of the apical to the basolateral membrane resistances for the rabbit urinary bladder while Wills and Clausen 1985 independently measured the resistance of the tight junctions using either antibiotics or the sodium channel blocker amiloride Of interest is that the first equation demonstrates that the product of the capacitors divided by the sum is independent of the equivalent circuit model This value is called the effective capacitance and since in most epithelia Cy is greater than Ca by about a factor of 5 the effective capacitance is approximately equal to Ca This relationship has been used to measure the change in membrane surface area as a function of alterations in epithelial transport rate 32 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company APPENDIX References and recommended reading e Epithelial Transport A guide to methods and experimental analysis Edited by Wills N K Reuss L and Lewis S A Chapman amp Hall London 1996 e Clausen C Lewis S A and Diamond J M 1979 Biophysical Journal 26 291 318 e Crowe W E and Wills N K 1991 Pflugers Archives 419 349 357 e Lewis S A and Hanrahan J W 1989 Methods in Enzymology 192 632 650 e Lewis
25. esistance iii A computer with acquisition hardware and software A well chosen acquisition system can simultaneously display several data channels while digitizing and storing the data in memory for later viewing and analysis An advantage of using a computer interfaced with the current voltage clamp is that the data can be immediately analyzed providing feedback on the viability of the epithelium iv If a computer is unavailable then a 2 4 channel paper chart recorder and oscilloscope is necessary The chart recorder will provide a permanent record of the experiment and should have a pen speed fast enough to record changes in transepithelial voltage or current produced by the pulse generator An oscilloscope is required to display the time dependent changes in transepithelial voltage during a current pulse In addition the oscilloscope can be used to determine whether the current voltage clamp is stable i e it is not oscillating as well as the response time of the clamp e g is the current 17 EC 825A Rev 120924 1 WARN ER INSTRUMENTS A Harvard Apparatus Company or voltage pulse that is being passed square or does it have a finite rise time In the EC 825A the speed of the clamp i e rate of rise of the current pulse can be selected on the front panel A square current pulse is important when estimating epithelial surface area using capacitance measurements see page 26 18 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Ha
26. este ee etes rosse have oet eo restera vn peo Eee Nee ospi re PORE CE ease e ERE See oroso OE E Re eR Pe Re osise oS 21 From epithelium to headstage eese esses eene tnne en tne tenentes entes ernst ne nens 21 From the current voltage clamp to external monitors eese eene enne enn 21 Checking for asymmetries in the voltage electrodes eee e eee eee eee eee eerte eese ee setae et enaee 21 Recording modes ree terrere ee exeo ett Prata rare Ero Eee eroe ur ea esenea beboes obo roa beoe enia voa ras posevusnevseseos desesssnatoents 22 Warnina lights me T 23 Fluid ecrire NM torns srece stecen esere resepe senses oi cos eetris SSeS r eros ro oes esi T E OSS KE Es aS aries SSS 23 Analyzing the electrical properties of an epitheliuim ccscssssccssssccsssssssssssssccsssssssssssssesssssecees 24 Transepithelial voltage seti n Rt TR ene TO ado dre aah A E e 24 Transepithelial resistance 2 sr o e reet E ctr EE A E O a se RE Osa 24 SHOVE CLV CULL 6CUFTEnt ic hikes tae et eerte eve Pak e Les eu aE a aeter ete Babette veg apte ree aa Pc eia 25 Equivalent circuit analysis eee eee esee eee eene nnne i Ea tnnt teen enr ennn a 25 Determining series resistance and epithelial surface area eese eee 29 Determining series resistance and epithelial surface area eese eene 30 Measuring fluid resistance in the presence
27. f the associated green LED Timer B TIMER B is used to set the on time for either voltage clamp or current clamp modes as selected with the associated toggle switch TIMER B on condition is indicated by lighting of the associated green LED Rear panel description The instrument rear panel provides connections for power entry grounding control inputs and data outputs 05 0254 Stow Bow W Grounds line power connector and fuse Both circuit black and chassis green grounds are provided at rear binding posts The instrument is shipped with the two grounds J connected via a shorting bar If needed this BB 05 0254 Siow Blow shorting bar can be disconnected allowing gy separation of the grounds Separating circuit EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company and chassis grounds can in some cases result in a reduction of 60 Hz noise levels However experimentation will determine which approach is best suited for your particular set up The power cord connects to the power input module Verify that the 110 220 V selector switch is set for the correct operating voltage The fuse 3AG type regular blow used in the EC 825A will depend on the line voltage 1 2 amp for 100 or 130 VAC and 1 4 amp for 220 or 240 VAC Replacement should only be made with the proper ampere rating Timer section The rear panel TIMER E Ll section is comprised of fg SSPE QUE C TNPRQUR output BNC
28. fluid resistance toggle switch to the right e To set the channel for 0 100 Q operation slide the fluid resistance toggle switch to the left Connecting the EC 825A to Acquire amp Analyze The basic Acquire amp Analyze data acquisition system from Physiologic Instruments is comprised of a DataQ digitizer and the Acquire amp Analyze software Two 2 connecting cables are also supplied a DB25 25 pin D connector printer cable for connecting the DataQ to your computer and a DB37 ribbon cable for connecting the DataQ to an input device These components are all that s needed for setup if using an EasyMount multichannel amplifier If you re using the Warner EC 825A however then additional components are required namely a MODO8 A connection box and the associated cables Generally speaking you ll make connections from the amplifier to the MOD08 A and from the MOD08 A to the DataQ Hardware connections l Begin by installing the Acquire amp Analyze software and connecting the DataQ to your computer according to the instructions that came with that equipment 2 Next connect the MODOS8 A to the DataQ using the 34 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company supplied DB37 ribbon cable Use the left side DB37 input on the front of the DataQ The EC 825A connecting cable has a single DB15 connector on one end and two DB9 connectors on the other end Each DB9 is labeled as CHANNEL 1
29. ince the tissue is bathed by symmetric solutions For simplicity we have left out a series solution resistor Since parallel conductors add the transepithelial conductance 25 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Gt the conductance between M the mucosal solution and S the serosal solution is given by G 2G G Since resistance is the inverse of conductance Gi 1 Ri then the transepithelial resistance is given by E R R i RSR and from Ohm s law the current flow i around this circuit is E C R R Again using Ohm s law we find that the voltage difference across the epithelium Vn Vs Vi is equal to the current flow through the tight junction E R V iR f R R or through the cellular pathway R ER ER V E iR E da fy et R R R R R c Inspection of the above equations reveals three interesting features e The magnitude of the transepithelial potential is a function of both the cell and junction resistance and the magnitude of the cell voltage source e The term E R is equal to the short circuit current and is indeed equal to the current generating capability of the cells e At a constant Ee a decrease in Re will result in an increase in Vi Thus as Re approaches zero V approaches E Similarly as Rc increases towards infinity then Ri approaches Rj This last relationship can be derived and formalized in the following m
30. ions bathing both sides of the epithelium are identical Unequal Cl concentrations will result in an asymmetry voltage difference between the voltage measuring electrodes This asymmetry voltage will be summed into the transepithelial voltage yielding a voltage artifact i e the measured voltage will be high or low depending upon the ratio of mucosal to serosal Cl concentrations ii If the epithelium is sensitive to Ag or is bathed by solutions containing different Cl concentrations then agar bridges connected to either Ag AgCl electrodes or calomel electrodes must be used The advantage of the agar bridge Ag AgCl electrode combination compared to calomel electrodes is that they are small and easy to locate close to the epithelial surface Moreover they are inexpensive Agar bridge electrodes must be connected via a salt solution to either the Ag AgCl or calomel electrodes which are connected in turn to the EC 825A The same electrode configurations can be used for the current passing limb of the electronics Fabrication Sintered Ag AgCl pellet electrodes are commercially available from Warner Instruments Call our offices or see our catalog for our extensive selection An alternate choice is to use silver wire which has been chlorided by the user See Appendix for techniques on chloriding Ag wire Agar bridges are easily constructed by heating a mixture of 596 agar with 1M KCl w v While still hot the agar can be drawn into polyethylene
31. ions of the equation has been violated in this instance E is not constant Note however that the plot of G versus ls is reasonably linear even though the assumption of a constant E has been violated To use this method only the resistive conductive properties of the cellular pathway must change and the change must be sufficiently large to result in a significant change in the measured parameters i e Vi Ri Isc or Gi As a consequence this method is most profitably used on the so called tight epithelia since a change in the cellular resistance results in a significant change in Ri In leaky epithelia where Rj can be 10 fold lower than Re large perturbations in this case a decrease in Re must be produced to obtain a reliable measurable decrease in Ri 29 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Determining series resistance and epithelial surface area In this section we discuss how the series fluid resistance can be estimated the with the epithelium in the chamber as well as how to estimate the surface area of the epithelium First we must draw an equivalent electric circuit which describes the epithelium and the fluid resistance in terms of its resistive and capacitative properties In this circuit the fluid resistance and the tight junction are modeled as resistors while the apical and basolateral membranes of the epithelium are represented by a parallel arrangement of a resistor and capacit
32. itude of the current step will be equal to the fluid resistance A square current pulse the rise time of the current step must be less than 10 us must be used to perform this measurement The following protocol can be used to make this measurement e Set the MODE SELECTOR switch to c clamp e Set the CLAMP RESPONSE to fast e Connect the V MONITOR BNC to a high speed oscilloscope and make sure that the voltage signal is not being filtered by the oscilloscope amplifier 30 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company e Apply a square current step to the EXT CMD input BNC e Measure the magnitude of the initial voltage jump Since the V MONITOR output BNC has x10 gain divide the measured voltage by 10 e Divide the measured voltage by the magnitude of the applied current step The resulting resistance is equal to the fluid resistance Measuring epithelial surface area To estimate the epithelial surface area one must analyze the time dependent change in the membrane voltage The complete protocol for analyzing these voltage transients has been described in detail by Lewis and Demoura 1984 In brief the on voltage response to a square current pulse is digitized at 100 us point with a resolution of 0 05 mV and stored in computer memory This data is next converted to an off voltage response by subtracting the time dependent on voltage response from the pre stimulus voltage The absolute value of
33. ltmeter and resistance Unique Features Include Y Membrane Resistance Measurement Accurate resistance measurements are made with the membrane mounted in the chamber A low frequency bipolar signal is used to avoid polarization of the membrane ideal for monolayers Resistances up to 200 kQ are displayed on the meter Y Clamp Speed Selection up to 10 ps Three clamp speeds adjust recording conditions for a variety of applications In Fast mode preparations with low access resistance small tissues or monolayers can be clamped with speeds up to 10 us Typical Ussing chambers with larger tissues will use Medium or Slow modes for stable oscillation free clamping Y Independent Voltage and Current Commands Internal command controls are provided for both voltage clamp and current clamp modes v Watertight Headstage with Model Membrane The small compact headstage can be located close to the measurement site to keep input leads short for reduced noise pick up Internal circuits are protected against the invasion of corrosive salts by a watertight seal The model membrane simulates a preparation and provides a convenient tool for making operational checks of the instrument Y High Voltage Compliance The high voltage compliance 50 V of the EC 825A is important for studies of low resistance e g leaky epithelial cells and in applications where long leads in the current passing circuit produce a voltage drop which must be compensated Additio
34. nally high compliance aids in charging large membrane capacitances resulting in faster settling times and improved overall clamp performance Additional Features Include Y High CMR Differential voltage recordings are made with very high common mode rejection providing accurate measurements free from the effects of common mode potential changes of a noisy environment Y Onboard Timer Controller The EC 825A includes two event timers to provide cycle times up to 2000 seconds Times are set using 2 digit thumbwheel switches and 4 position range switches Once set the timer will free run eliminating the need for a computer or other external device to control the experiment Y External Instrument Control The clamp can be operated by an external programmer lab timer or by a computer Logic control of clamp mode and clamp command levels is possible as well as simultaneous mixing of external linear commands EC 825A Rev 120924 1 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 rather than complexity we welcome any feedback you may wish to provide gt Warner Instruments product numbers a
35. nce between the voltage measuring electrodes is composed of a series combination of the resistance of the epithelium and the resistance of the fluid bathing the tissue this latter resistance is sometimes called the series resistance To determine the epithelial resistance the fluid resistance must be measured and subtracted from the total resistance Moreover to voltage clamp a tissue at a specified value then the fluid resistance must be compensated for The fluid resistance can be measured and set using one of the following protocols Method A e Connect all electrodes to the chamber and fill with the appropriate Ringers solution in the absence of an epithelium e Switch the HEADSTAGE SELECTOR switch to on e Set the METER SELECTOR switch to voltage e While depressing the PUSH TO ADJUST button in the FLUID RESISTANCE control block adjust the POTENTIOMETER until the voltage reading on the meter reaches O mV 23 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Method B e Attach the electrodes and fill chamber as described above e Switch the HEADSTAGE SELECTOR switch to on e Switch the MODE SELECTOR switch to MEMBRANE RES mode and read the series resistance from the meter e Set the FLUID RESISTANCE POTENTIOMETER to the value read from the meter in the last step Finally see page 28 for a method of measuring fluid resistance with the epithelium in place i e the fluid resistance adjustment need
36. new clamping voltages should be displayed on the panel METER e Return the VOLTAGE COMMAND TOGGLE switch to off 19 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Checking clamp current on panel meter e Set the METER MODE switch to current The meter now displays the current necessary to clamp the membrane to zero volts Fluid Resistance compensation e Set the MODE SELECTOR switch to amplify e Set the METER MODE to voltage e Set the HEADSTAGE SELECTOR switch to test and the TOGGLE switch to fluid resistance e Press the PUSH TO ADJUST button in the FLUID RESISTANCE control block The LCD meter should display a non zero value e While depressing the PUSH TO ADJUST button advance the FLUID RESISTANCE POTENTIOMETER until the meter again reads zero The 10 turn dial on the potentiometer should read approximately 47 Q the value of the dummy fluid resistor e Switch the METER MODE switch to current e Press the PUSH TO ADJUST button in the FLUID RESISTANCE control block to check the amplitude of the fluid resistance test current it should be set for 25 uA 20 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company OPERATION Procedures are presented for using this experimental set up to measure the basic electrical properties of epithelia including the transepithelial potential difference the transepithelial resistance short circuit current and capacitance C
37. not be performed before the start of an experiment Analyzing the electrical properties of an epithelium After mounting the epithelium into a chamber connecting the current voltage leads to the amplifier and eliminating asymmetries in the voltage measuring electrode three basic properties of the epithelium can be measured These are the transepithelial voltage the transepithelial resistance and the short circuit current Isc a measure of the net active ion transport across the epithelium Transepithelial voltage The first measurement is straightforward and is simply read from the panel meter or from a chart recorder or oscilloscope One must remember to make sure that there is no asymmetry potential between the voltage measuring electrodes Since the transepithelial voltage is measured differentially one must know which one of the two voltage measuring electrodes is considered zero or ground Transepithelial resistance The transepithelial resistance can be measured by either passing a current across the epithelium AI and measuring the resultant voltage change AV under current clamp mode or by clamping the epithelium to a new voltage AV and measuring the change in current AI under voltage clamp mode The resistance is then calculated using Ohm s law which simply states that the resistance is equal to the change in the transepithelial voltage divided by the change in the transepithelial current yeas ODA AI t R
38. onnecting the EC 825A This section describes the steps involved in connecting the EC 825A to an epithelium From epithelium to headstage Start with the instrument power off Set the MODE SELECTOR switch to amplify Set the HEADSTAGE SELECTOR switch off Place the voltage measuring and current passing electrodes into the chamber Connect the electrodes to the HEADSTAGE INPUTS using the provided color coded leads From the current voltage clamp to external monitors The EC 825A is a completely self contained unit However a permanent record of your data can be exported to a paper chart recorder oscilloscope or computer via the clamp BNC outputs V MONITOR and I MONITOR in the CHANNEL control block on the rear panel Checking for asymmetries in the voltage electrodes Prior to beginning an experiment asymmetries in the voltage measuring electrodes must be compensated Set the HEADSTAGE SELECTOR switch to off Select amplify on the MODE SELECTOR switch Set the METER MODE toggle to voltage Place both voltage measuring electrodes HEADSTAGE V1 and V2 INPUTS into the same bath Turn the HEADSTAGE seLector switch to on If the voltage reading on the meter is non zero then use the INPUT OFFSET POTENTIOMETER to adjust the voltage reading to zero As a rule of thumb if the offset voltage is greater than 10 mV then the electrodes must be replaced and the offset readjusted Set the HEADSTAGE SELECTOR switch to off
39. or The figure below shows this circuit the tight junctional resistance is not included since it is infinite and outlines the response of the circuit to a square current pulse The characteristic equation describing the voltage response to the current input signal is also shown Note that the voltage response to a square current waveform is time dependent Consequently this response can be analyzed in the time domain V t OR R e191 R l e 2202 Figure 4 Response of a simple epithelial equivalent circuit to a square current input and the generalized equation which describes the voltage output The output response is shown as two easily discernible exponential traces In this example the value of the two time constants R C and R2C2 were selected to be greatly different If the time constants are approximately equal this circuit will show a single exponential response similar to the single R with series resistor The voltage V current i and time t scales are in arbitrary units This figure demonstrates the response through a series resistor and two parallel resistor capacitor combinations arranged in series This response is characterized by an initial voltage jump equal to the fluid resistance followed by the sum of two hyperbolic curves each curve being a function of an individual Measuring fluid resistance in the presence of an epithlial membrane The magnitude of the voltage jump shown in Figure 4 divided by the magn
40. plifier speed of the current and voltage circuits series resistance compensation and internal pulse generation Compliance voltage of the current passing amplifier defined as the maximum voltage output of the amplifier is important if agar leads are used as part of the current passing circuit Some clamps use amplifiers which have a compliance voltage of 10 volts If the total resistance of the current passing circuit is 10 kQ this means that the maximum current the clamp can pass is 1 0 mA Although this current level is sufficient for high resistance tight epithelia it most likely will be marginal for low resistance leaky epithelia or for epithelia whose resistance has been decreased due to an experimental maneuver The EC 825A uses a 110 V current passing amplifier making this an instrument of choice for leaky epithelia The EC 825A has outputs which can be easily connected to chart recorders oscilloscopes or computers Minimum equipment requirements are i A current voltage clamp amplifier This is essential to record the transepithelial voltage the transepithelial resistance and the short circuit current Is ii A pulse generator This device is necessary to either pass a current across the epithelium and record the change in the transepithelial voltage or to voltage clamp the epithelium and record the current passed to maintain that voltage Either of these two measuring schemes allows the calculation of the transepithelial r
41. re presented using bold type gt References to instrument panel operational sections are specified using ITALICIZED UNDERLINED CAPS e g CHANNEL 1 TIMER A References to instrument panel control blocks within operational sections are specified using UNDERLINED SMALL CAPS e g METER CLAMP COMMANDS References to controls within a block are specified using NON UNDERLINED SMALL CAPS e g MODE SWITCH TIMER RANGE References to control settings are specified using italic type e g Amplify 100 mV gt Special comments and warnings are presented in highlighted text Any other formatting should be apparent from context EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company CONTROL DESCRIPTION The following is a description of the operating controls inputs and outputs located on the front and rear panels of the instrument The EC 825A front panel is comprised of two channel sections separated by a timer section Each channel is subdivided into control blocks termed OFFSET CLIPPING METER MODE COMMAND FLUID RESISTANCE and CLAMP RESPONSE The timer section is divided into TIMER A and TIMER B control blocks Channel 1 and 2 control blocks Offset The toggle switch selects input polarity or or off no offset The ten turn control provides input offset adjustment from 0 120 mV Clipping The HEADSTAGE for the associated channel connects to the EC 82
42. rvard Apparatus Company Quick test Each EC 825A headstage contains a model membrane which can be made functional by selecting the test position eee on the HEADSTAGE SELECTOR switch R6 m 100K m INPUT V1 Initial control settings For each channel ps 47 e Set the HEADSTAGE SELECTOR S1A switch to off zi Si m e Set the METER MODE switch to gn S8 SK 15V E voltage AAA L mmi T tour ELINPURVZ e Set the MODE SELECTOR switch to E amplify 2 e C CURRENT 12 e Set the COMMAND polarity and E 8 INPUT OFFSET switches to off Schematic of Model Membrane After the instrument has warmed up the digital voltmeters should show a potential close to O mV Completion of the following procedures verifies the proper operation of the voltage clamp Monitoring model membrane voltage e Set the HEADSTAGE SELECTOR switch to test and the TOGGLE switch to Vmem The digital panel meter should show a potential in the neighborhood of 100 10 mV depending on the condition of the battery inside the headstage Checking zero voltage clamp e Change the MODE SELECTOR switch to V clamp The panel meter should read 0 V The membrane is now clamped to zero volts Check voltage clamp command e If the VOLTAGE COMMAND TOGGLE switch is moved to the left or right or selected the 10 turn dial becomes active and the instrument will clamp the membrane at voltages other than zero volts These
43. s These include i Temperature control The chamber must have provisions for controlling the temperature of the mucosal and serosal bathing solutions and for aerating both solutions with the gas mixture of choice ii Damage control The chamber must have a design which minimizes damage to the cells which contact the inner circumference of the hemi chamber If not protected against such damage termed edge damage will result in a measured epithelium resistance which is lower than the native tissue iii Support The epithelium must be supported on one side by a rigid but permeable structure This reduces epithelial stretch and the possibility of cell or tight junction damage iv Voltage electrodes The voltage measuring electrodes must be placed as close as possible to the epithelium This will reduce the magnitude of the solution series resistance which if large can compromise the ability to precisely voltage clamp the epithelium v Current electrodes The current passing electrodes must the placed in the rear of the chambers and as far as possible from the epithelium This will assure a uniform current density across the epithelium A non uniform current density will result in an overestimate of the epithelial resistance vi Solution changes Bathing solutions must be easily and rapidly changed without interrupting electrical measurements and without altering the electrical properties of the epithelium 14 EC 825A Rev 120924
44. steady state current can be varied in magnitude and polarity within the COMMAND control block The voltage measured in this mode is produced by the active transport of ions by the epithelium plus the current applied across the epithelium by the current passing amplifier An advantage of the zero current clamp mode that in the presence of Ag AgCl wires the silver contamination of the bathing solutions is minimized Resistance of the membrane In this mode the EC 825A automatically determines the membrane resistance by passing a bipolar current pulse of 10 uA 0 2 KQ range or 1 uA 0 200 kO range The resistance is read directly from the panel meter in units of O or KQ e g 103 Q If the meter displays all zeros then select the Q setting via the associated toggle in the MODE section If the meter is blank except for a 1 on the far left then select the KO setting If the meter s reading does not change then either the resistance of the tissue is larger than 50 kQ or there is a high resistance connection between the headstage and the current or voltage electrodes in the chambers External timer This allows the operator to remotely select between current clamp TTL high or voltage clamp TTL lou modes using either a switching box not provided or a computer input connected to the EXT TIMER IN BNC on the rear panel 22 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company In this mode a TTL low signal at the GATE IN
45. tubing using a syringe or vacuum line Since the polyethylene tubing is opaque it is convenient to add a dye e g methylene blue to the agar KCl solution This allows the visualization of any discontinuity in the agar bridge which can cause an overload lights and audio signal from the input and or clamp amplifier P EEA U If the interface resistance is too large it will limit the current passing capacity of the electronic equipment Ideally use of a Ag AgCl sheet or wire coil in the rear of each hemi chamber is sufficient to assure a uniform current density across the epithelium However if the tissue is sensitive to trace levels of Ag a wide bore agar bridge placed at the rear of each hemi chamber will be required to make a connection between the electrodes and 16 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company solution If this technique is necessary then every effort should be made to keep the length of the bridge short and to fill the bridge with a low resistance solution to maximize the current passing capability of the electronic equipment A disposable pipette tip partially filled with agar back filled with a low resistance solution 1M KCl and coupled to the electronic equipment with a Ag AgCl wire or sintered pellet works very well Electronics Four important features of an epithelial clamp are described in this section They are compliance voltage of the current passing am
46. ue Isc is measured by voltage clamping the epithelium to O mV and reading the applied current from the panel meter When performing such measurements it is essential that one compensates for the series resistance Rs by using the FLUID RESISTANCE control block on the EC 825A Equivalent circuit analysis We now introduced the concept that an epithelium can be modeled as an electrical circuit composed in the most simple case of a resistor and a voltage source The justification for this electrical approach is that many epithelia produce a spontaneous potential even when bathed on both sides with identical solutions Therefore this potential can be represented as a voltage source Since an epithelium is capable of restricting resisting the movement of ions between two compartments it has resistive properties Therefore it can be represented as a resistor Below is the most simple equivalent circuit of an epithelium based on its morphology In the equivalent circuit shown to the right the cells of the epithelium are represented by a resistor Re in series with a voltage source E lt while the parallel tight junctions are represented by a simple resistor This voltage source also called the cellular electromotive force EMF is a complex function of the conductive properties of the cell membranes and the composition of the ions present in the bathing solution as well as in the cell interior No voltage source is present in the junction s
47. urrent 100 106 uA In the Clamp Mode section select Current Clamp and hit Apply Verify that the amplifier is now in current clamp Verify that the meter reads voltage 102 mV and current 0 uA 35 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company 13 In the Clamp Mode section select Open Circuit and hit Apply Verify that the amplifier remains in current clamp Verify that the meter reads voltage 102 mV and current 0 uA 14 In the Clamp Mode section select Voltage Clamp Click on Apply and exit the Pulse Setup dialog box by hitting OK 15 Open the Reference Setting Dialog Box Acquire Reference Click on the Reference button and allow the algorithm to fill the table with values If you see all white boxes all yellow boxes or a combination of white and yellow boxes then the instrument and software are communicating properly If you see any red boxes with values greater than 1 0 then contact Warner tech support for assistance This completes the Warner specific setup instructions for Acquire amp Analyze Please refer to the Acquire amp Analyze documentation for how to use the software to set up and run experiments 36 EC 825A Rev 120924 1 Specifications Headstage Input Impedance 1x10 0 shunted by 6 pF Input Voltage 41 5 V maximum Common Mode Voltage 10 V maximum Common Mode Rejection 110 dB 60 Hz Leakage Current 20 pA max Offset Voltage Range 120 mV Voltage Clamp
48. used to compensate for any voltage drops due to the intrinsic resistance of the bathing solutions A 25 uA current this value can be adjusted internally is injected at the headstage 11 OUTPUT The measurement or adjustment is made by depressing the PUSH TO ADJUST button and turning the ten turn dial until zero voltage is displayed on the digital meter The fluid resistance is read from the dial 0 100 Clamp Response CLAMP RESPONSE Selects fast med or slow clamp speeds This control is active in both current and voltage clamp modes EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company Timer control blocks The TIMERS are used to program the on and off times of either one or both channels of the EC 825A TIMER controls can be activated internally by selecting Int Timer in the MODE control block and externally by selecting Ext Timer in the MODE control block When in Ext Timer mode a TTL high voltage at the EXT TIMER IN BNC on the rear panel selects TIMER A and a TTL low voltage selects TIMER B Time durations are set using two thumb wheel digits and a multiplier switch Times can be set from 10 ms to 990 seconds in 4 ranges x10 ms x100 ms x1 sec and x10 sec Timer A The TIMER A section is used to set the off time or zero clamp time for either voltage clamp or current clamp modes as selected with the associated toggle switch TIMER A on condition is indicated by lighting o
49. ve with respect to a solution containing NaCl 0 9 or KCl 3M and passing a current through the electrode at a rate of 1 mA cm of surface area for 10 15 seconds or until adequately plated a 1 cm length of 1 mm diameter wire will require approximately 0 3 mA The color of a well plated wire should be purple gray Periodic reversal of the polarity while plating the electrode tends to yield a more stable electrode When electroplating a previously plated wire you may find that it does not plate evenly Complete removal of the residual silver chloride is usually necessary to effect a uniform coat Before making the wire positive to the chloriding solution reverse the polarity for 33 EC 825A Rev 120924 1 WARNER INSTRUMENTS A Harvard Apparatus Company 5 to 10 seconds to remove any remaining chloride that might be left in pits on the wire Then proceed as described above Adjusting the fluid resistance measurement range Unless otherwise specified the FLUID RESISTANCE measurement range is factory set for 0 100 Q If required the FLUID RESISTANCE range can be extended to 1 KQ by setting the fluid resistance toggle switch on the main circuit board Each channel must be adjusted separately The procedure to make this adjustment is as follows e On the top of the rear panel remove the two Philips head screws holding the top cover in place and remove the cover by sliding it back e To set the channel for 0 1 KQ operation slide the

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