EC-800 User`s Manual (120924)
Contents
1. 800LV Manual Rev 050815 Specifications Headstage Input Impedance Input Voltage Common Mode Voltage Common Mode Rejection Leakage Current Offset Voltage Range Voltage Clamp Internal Clamp Potentiometer External Command External Command factor Current Clamp Clamp Potentiometer External Command Command Factor Speed Compliance EC 800 EC 800LV Resistance Fluid resistance Compensation range Membrane Resistance Measurement Ranges Panel Meter Voltage Range Current Range Power Requirements Physical Dimensions EC 800 and EC 800LV Headstage Shipping Weight Warranty Operating Conditions Equipment is intended to be operated in a controlled laboratory environment WARNER INSTRUMENTS A Harvard Apparatus Company 1 x 10 Q shunted by 10 pF 1 5 V maximum 13 V maximum 120 dB 60 Hz 500 pA max 120 mV 100 mV with 10 turn control 1 V 1 mV 10 mV applied 1 mA 10 mA 1 uA 10 mV applied 10 us measured with model membrane 120 V 15 V 0 100 standard 0 1 KQ optional made with a 2 Hz bipolar constant current square wave 0 2 KQ injected current 10 pA 0 200 KQ injected current 1 pA 3 5 digit LCD 200 mV maximum 2000 uA maximum 100 130 VAC or 220 240 VAC 50 60 Hz 17 8 cm H x 42 cm W x 25cm D 7 7 cm H x 7 7 cm W x 5 cm D 9 1 kg Three years parts and labor Temperature 0 40 C Altitude sea level to 2000 m Relative humidity 0 95 302. Directive to its products and will comply with any changes as they apply Do Not Dispose Product with Municipal Waste Special Collection Disposal Required 33 33
3. EC 800 Rev 050815 Certifications 31 Declaration of Conformity CE MARKING EMC Application of Council Directive 89 336 EEC Standards To Which Conformity Is Declared Manufacturer s Name Manufacturer s Address Equipment Description Equipment Class Model Numbers EN55022 Class A 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 Warner Instruments LLC 1125 Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Instrument Amplifier ITE Class A EC 800 EC 800LV I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Connecticut USA Gab gri Full Name Burton J Warner Signature Position President 31 EC 800 Rev 050815 32 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 EC 800 EC 800LV I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Conn
4. EC 800 however then additional components are required namely a MODO08 A connection box and the associated cables Generally speaking youll 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 MODOS A to the DataQ using the supplied DB37 ribbon cable Use the left side DB37 input on the front of the DataQ 3 The EC 800 connecting cable has a single DB15 connector on one end and a single DB9 connector on the other end Connect the DB15 to the associated port on the rear of the EC 800A and the DB9 to a CLAMP input on the MOD08 A 28 EC 800 EC 800LV Manual Rev 050815 WARN ER INSTRUMENTS A Harvard Apparatus Company 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 input on the MODO8 A e g CLAMP 1 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 A
5. R left panel and Gi versus I 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 Fj 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 and J E G or G ada i E Substituting gives I G G E Using this equation a plot of the transepithelial conductance Gi 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 resistance or conductance Experience has shown that a plot of Vi vs Ri is more sensitive to changes in either Rj or E than a plot of G vs Is 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 an
6. Start with the instrument power off e Set the MODE SELECTOR switch to Amplify e Set the HEADSTAGE SELECTOR switch off e Place the voltage measuring and current passing electrodes into the chamber Connected the electrodes to the HEADSTAGE inputs using the provided color coded leads From the current voltage clamp to external monitors The EC 800 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 VOLTAGE MONITOR and CURRENT MONITOR in the FLUID RESISTANCE control block Checking for asymmetries in the voltage electrodes Prior to beginning an experiment asymmetries in the voltage measuring electrodes must be nulled compensated e Set the HEADSTAGE SELECTOR switch to off e Select amplify on the MODE SELECTOR switch e Set the METER MODE toggle to voltage e Place both voltage measuring electrodes HEADSTAGE V1 and V2 inputs into the same bath e Turn the HEADSTAGE selector switch to on e 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 e Set the HEADSTAGE SELECTOR switch to off e Place the HEADSTAGE voltage measuring electrodes into their respective chambers 16 EC 800 EC 800LV Manua
7. ena 16 EC 800 EC 800LV Rev 120924 From epithelium to headsiage eee ed ette eee tenete edn eei reed eno erbe eee e ERE e Rede RR e USTO Skia 16 From the current voltage clamp to external monitors eee esee eene enne 16 Checking for asymmetries in the voltage electrodes eee esee ee esee eene eren eee en setate sten as etna 16 Icon eL EASO reosta 17 M PYanunALU ce 18 Fluid r sistance 5 ccccssiesicascnssessesseasesassoscovasusaassssdcnesssscesenseFacseseossoascasessccnsesdeassassoasesassesceas bos SSpS Risorsa 18 Analyzing the electrical properties of an epithelium ssesssesssesssesssecsecesocesocesocesocssoossooseoosssosssoseso 18 TPransepithelial voltage e eue t eit eet e AAEE RE E E EERE HTC E HH Rae 19 Transepithelial resistance ue e inte van Wd eene eh te tees eee tating ven petere er ves e a san cats pee adea 19 Short circuit CUT Tell see er ER EE RR EE EP WEE ea NA Debe MAE PESE Eod e nin 19 Equivalent circuit analysls eei e ie t e en eet etg ete ertet eet egre a tes 19 Determining series resistance and epithelial surface area eese 24 EXuuolb beee XX 27 References and recommended reading 4 eee eee eee eee e ee esee seen sette setas eee en setate setae s eaae eaa 27 Chloriding silver WIPES eiie eee eoo eese poen eee eo ie oth a ego tk Coa nae tio eee ues eun bassa ee oues ovn stabe eres ee
8. fill chamber as described above e Switch the HEADSTAGE SELECTOR switch to on e Switch the MODE SELECTOR switch to R MEMBRANE 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 23 for a method of measuring fluid resistance with the epithelium in place i e the fluid resistance adjustment need 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 18 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company 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 transepit
9. EC 800 EC 800LV Rev 120924 WARNER INSTRUMENTS A Harvard Apparatus Company Warner Instruments Epithelial Voltage Clamp Models EC 800 amp EC 800LV Warner Instruments 1125 Dixwell Avenue Hamden CT 06514 800 599 4203 203 776 0664 203 776 1278 fax EC 800 EC 800LV Rev 120924 Table of Contents DESCRIPTION Pe M SEN 4 NOMENCLATURE inest Em 5 CONTROL DESCRIPTION 6 Front panel control blocks 5 cee er Ice rete esee ee Gore re ero s ean Go oe ee ena asset co Sero KASS AVENA Se coena ENARA Aie 6 Input Offsetz iet enisi nte etii titm ed ici tseie eerte 6 Fluid Resistance and monitors esses essen nennen enne EA per iA nene Ee entere nnne sr aa a 6 MO etre terere epi bot inae tive ote e ae eot eue TREE dg e br toros E Ee ERR PORE 6 Commands suo atas deas vette dide seed e Goad d eviter dre O dedicate redde ease ewes n ara c 8 Rear panel CESCHIPUOMN cssiciscccdsssccssoestsceccsancoosescesevecsigcesseedaseseesetestesacesceccedssesebesasceecedescessensdenaseeesceseecs 8 Line power connector and fuse sssseesenseeseessersseessersserssersserssessssssesssessseesseesstesstesstesseessressessseest YO DOT i eie a re TERES PERRA E E D FEE aT APNE TE FEE Pe RR See MAE TERREA 8 DOLE PU RTT 9 Additional components and comments sesssesssesssecesecssccesccesocesocesocssocescossoossoossoosssoss
10. STAGE l1 output This is a true amplify operation C Clamp Selects current clamp mode R Membrane Selects membrane resistance measurement mode A constant current 2 Hz square wave is injected and the resultant voltage is measured across the membrane The calculated resistance is displayed on the meter Ext Timer External timer mode A TTL signal applied to the EXTERNAL TIMER IN BNC will switch the amplifier between current clamp TTL high and voltage clamp TTL low modes The CLAMP RESPONSE push button selects fast med or slow clamp speeds This control is active in both current and voltage clamp modes The R MEMBRANE RANGE push button selects the resistance measurement range 2 or kQ used when R Membrane is selected on the MODE SELECTOR switch A 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 The membrane resistance read on the panel meter which scales automatically with the range selected Two input BNC s are included within this control block and are described below EXT SIGNAL INPUT An external voltage or current clamp command input Applied current or voltage commands are summed with their respective counterparts in the COMMANDS control block In voltage clamp mode the ratio of EXT SIGNAL INPUT voltage to applied clamp voltage is 0 01 V mV In current clamp mode the ratio of EXT SIGNAL INPUT voltage to applied clamp current is 10
11. age 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 Ag contamination of the bathing solutions is minimized Resistance of the membrane In this mode the EC 800 automatically determines the membrane resistance by passing a bipolar current pulse of 10 uA 0 2 KQ range or 1 pA 0 200 kQ range The resistance is read directly from the panel meter in units of O or kO e g 103 Q If the meter displays all zeros then select the Q setting by pushing the R MEMBRANE RANGE button in the MODE control block If the meter is blank except for a 1 on the far left then select the kKQ 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 low modes using either a switching box not provided or a computer input connected to the EXTERNAL TIMER IN BNC In this mode a TTL low signal at the GATE INPUT BNC instructs the EXTERNAL TIMER IN BNC to select between the values set for voltage or current clamp modes By comparison a TTL high signal at the GATE INPUT BNC instructs th
12. ard Apparatus Company Checking clamp current on panel meter e Set the METER MODE switch to current uA The meter now displays the clamp 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 mV e Set the HEADSTAGE SELECTOR switch to test and the HEADSTAGE 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 15 EC 800 EC 800LV Manual Rev 050815 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 Connecting the EC 800 This section describes the steps involved in connecting the EC 800 to an epithelium From epithelium to headstage e
13. are specified in italic type e g Amplify 100 mV Input connections to the headstage are printed in a bold type e g 11 output V2 input Special comments and warnings are presented in highlighted text Any other formatting should be apparent from context EC 800 EC 800LV Manual Rev 050815 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 Front panel control blocks The front panel is comprised of four labeled control blocks termed INPUT OFFSET FLUID RESISTANCE MODE and COMMANDS A fifth unmarked section is termed the METER control block Input Offset The toggle switch selects input polarity or or off no offset The ten turn control provides input offset adjustment from 0 120 mV The HEADSTAGE connects to the amplifier in this control block Fluid Resistance and monitors This control block is used to compensate for any voltage drops due to the FLUID RESISTANCE intrinsic resistance of the bathing solutions A 25 pA current this value PUSH TO ADJUST a VOLTAGE CURRENT MONITOR MONITOR x10 10mV yA Monitors Two output BNC s are included in this control block for communicating amplifier can be adjusted internally is injected at the HEADSTAGE l1 output The measurement adjustment is made by depressing the PUSH TO ADJUST button and t
14. 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 employed 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 B 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 Electroplating Electroplating a silver wire with chloride is achieved by making the wire positive 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 5 to 10 seconds to remove any remaining chloride that might be left in pits on the wire Then proceed as
15. ature 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 10 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company vi Solution changes Bathing solutions must be easily and rapidly changed without interrupting electrical measurements and without altering the ele
16. ch 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 assumptions of the equation has been violated in this instance E is not constant Note however that the plot of Gi 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 R In leaky epithelia where Rj can be 10 fold lower than R large perturbations in this case a decrease in R must be produced to obtain a reliable measurable decrease in R 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 Re 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 23 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Determining series resistance and epithelial surface a
17. ctrical properties of the epithelium 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 bndge m Solution inlet m Current electrode m Agar bridge s Solution outlet s Solution inlet s Plexiglas insert serator outflow w Current electrode s a Teflon dish ps 4 i Silicone spacers Stainless 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 see ii Right this chamber is designed to be us
18. d the junctional resistance and is thus more sensitive to a change in either parameter This is illustrated below Figure 3 next page 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 22 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Three approaches have been used to alter R in a controlled manner These are increasing the cell membrane 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 since there is low chloride in the bathing solution cell swelling due to KCl influx is minimized and secondly since the Figure 3 A plot of V vs R left panel and Gi vs ls right panel In this example the cell membrane resistance was decreased using gramicidin D In addition the mucosal solution was selected su
19. described above 27 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company 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 jumpers on the main circuit board The procedure to make this adjustment is as follows e Remove the two Philips head screws holding the top cover in place and remove the cover e Locate the jumper blocks labeled W1 W8 W1 W4 are located on a single block next to the piggy backed circuit board W5 W6 and W7 W8 are located on two jumper blocks located towards the front of the main circuit board e To set the channel for 1 kO operation un jumper W1 W3 W5 and W7 and jumper W2 W4 W6 and W8 e To set the channel for 100 Q operation jumper W1 W3 W5 and W7 and un jumper W2 W4 W6 and W8 Connecting the EC 800 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
20. displayed for any selection within the MODE control block with the exception of R MEMBRANE mode In R MEMBRANE mode the meter reads the membrane resistance in either Q or kO as selected by the R MEMBRANE RANGE push button in the MODE control block Rear panel description The instrument rear panel provides several connections for control input and data output Line power connector and fuse The power cord connects to the power connector Check the serial number tag on the rear panel to be sure that the instrument is wired for the operating voltage used The fuse 3AG type regular blow used in the EC 800 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 VO port This 9 pin connector allows the EC 800 to be connected for computer operation Pin designations are 1 V MONITOR x 10 5 EXTERNAL TIMER IN 2 I MONITOR 6 7 amp 8 CIRCUIT GROUND 3 EXT SIGNAL INPUT 9 no connection 4 GATE INPUT EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Gate input Used to control the behavior of the EXTERNAL TIMER IN input BNC in the MODE control block A TTL low signal at the GATE INPUT BNC instructs the EXTERNAL TIMER IN BNC to select between the values set for voltage or current clamp modes By comparison a TTL high signal at the GATE INPUT BNC instructs the EXTERNAL TIMER IN BNC
21. dvanced Settings button Set the Instrument Type to EC 800 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 13 14 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 current 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 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 In the Clamp Mode section select Voltage Clamp Click on Apply and exit the Pulse Setup dialog box by hitting OK 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 29 EC 800 EC
22. dware 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 13 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company is not oscillating as well as the response time of the clamp e g is the current or voltage pulse that is being passed square or does it have a finite rise time In the EC 800 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 Quick test The EC 800 headstage contains a model membrane which can be made functional by s
23. e EXTERNAL TIMER IN BNC to select for zero current clamp for current clamp mode or zero voltage clamp for voltage clamp mode 17 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company 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 resistance 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 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 toggle 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 0 mV Method B e Attach the electrodes and
24. e noe Un iseni oou 27 Adjusting the fluid resistance measurement range 4 eee eee ee ee en eee ete etta sesta sete eo sette sten assa 28 Connecting the EC 800 to Acquire amp Analyze eee eere eerte ee ee ee eee te sten nest ense ttes etas esten ae eaa ae 28 Hardware connections e etae tette eet ides oe ARR ELMAR e EI Lak badd ides Hoo PL Re AEE e ETE Eat ped EL Ro eoo bake edd Rete 26 Software Config Urati ON i terree dee uude mayores ee eR T en ee e aN ev EE XAR d 29 Musei 30 LEO WEE DCTLUICSEDTEERTURERE TC M 31 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company The EC 800 and EC 800LV Epithelial Voltage Clamps from Warner Instruments provide accurate measurements of transepithelial voltage short circuit current and membrane resistance Important features include fluid resistance compensation membrane resistance readout high voltage compliance and small water tight headstages Operating modes include voltage clamp current clamp voltmeter and resistance Unique Features Include Y Membrane Resistance Measurement Accurate resistance measurements are made with the membrane mounted in the chamber The measurement is made using a low frequency bipolar signal to avoid polarization of the membrane ideal for monolayers Resistances up to 200 kO are displayed on the me
25. ecticut USA Signature Gab gri Full Name Burton J Warner Position President 32 EC 800 Rev 050815 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 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
26. ed 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 11 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Electrodes Electrodes are an essential component of any electrophysiogical 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 pass a transepithelial current Although it may seem trivial careful choice of 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 solutions bathing both sides of the epithe
27. ed to measure the change in membrane surface area as a function of alterations in epithelial transport rate 26 EC 800 EC 800LV Manual Rev 050815 WARN ER 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 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
28. electing the test CURRENT 11 position on the HEADSTAGE SELECTOR switch Initial control settings e Set the HEADSTAGE SELECTOR switch to off e Set the METER MODE toggle switch to voltage mV e Set the MODE SELECTOR switch to amplify INPUT V2 FLUID RESISTANCE lt e Set the polarity switches for INPUT OFFSET and the COMMAND controls VOLTAGE and CURRENT to their respective off positions Schematic of Model Membrane CURRENT 12 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 On the HEADSTAGE set the SELECTOR switch to test and the TOGGLE switch to Vmem The digital panel meter should show a potential in the neighborhood of 100410 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 or is selected on the COMMAND VOLTAGE toggle switch the 10 turn dial will voltage clamp the membrane at voltages other than zero volts These clamp voltages should be displayed on the panel METER e Return the COMMAND VOLTAGE toggle switch to off 14 EC 800 EC 800LV Manual Rev 050815 WARN ER INSTRUMENTS A Harv
29. esistance is equal to the fluid resistance MEASUREING 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 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 equivalen
30. helial 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 Br t meas AI 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 and the series resistance of the solution R 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 Ri Rmeas Rs the EC 800 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 correcti
31. high compliance For these applications the EC 800LV 15 V offer both a lower cost and a safer environment for the membrane 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 External Instrument Control The clamp can be operated by an external programmer lab timer or computer Logic control of clamp mode and clamp command levels is possible as well as simultaneous mixing of external linear commands EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company NOMENCLATURE Text conventions This manual refers to amplifier controls at three functional levels 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 gt Warner Instrument product numbers are presented using bold type References to instrument panel control blocks are specified using UNDERLINED SMALL CAPS e g METER CLAMP COMMANDS References to specific controls within a block are specified using NON UNDERLINED SMALL CAPS e g MODE SWITCH TIMER RANGE References to individual control settings
32. ince 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 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 12 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company 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 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 amplifier speed of the current and voltage circuits
33. is equipped with a built in model membrane allowing for a rapid and convenient instrument test See page 14 for test procedure EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Connecting the headstage The headstage connects to the instrument via an 8 pin DIN connector in the INPUT OFFSET control block To reduce noise the headstage should be placed as close to the test site as possible 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 features These include i Temper
34. ithelial 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 Ata constant E a decrease in R will result in an increase in V Thus as R approaches zero Vv approaches Eec Similarly as R increases towards infinity then Ri approaches Rj This last relationship can be derived and formalized in the following manner E R i V R rearranging yields E V t c c C and R T rearranging yields R R n LL R R R c j substituting the above two equations yields LA 1 E R If a perturbation which changes only the cell resistance is performed a plot of the resulting paired values of V and R during this perturbation yields a linear double intercept in which both Rj and E can be determined see Figure 2 next page 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 E 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 21 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company z a 5 20 3 s ape Lee aA IRF res b Figure 2 Plot of Vi versus
35. l Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company e 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 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 COMMANDS 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 Amplify In this mode the current passing amplifier is disconnected from the current passing electrodes HEADSTAGE I1 and l2 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 SIGNAL INPUT BNC will not be passed across the epithelium 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 steady state current can be varied in magnitude and polarity within the COMMANDS control block The volt
36. le 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 resistor capacitor network MEASURING FLUID RESISTANCE IN THE PRESENCE OF AN EPITHLIAL MEMBRANE The magnitude of the voltage jump shown in Figure 4 divided by the magnitude 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 VOLTAGE MONITOR BNC to a high speed oscilloscope and make sure that the voltage signal is filtered by the oscilloscope amplifier e Apply a square current step to the EXT SIGNAL INPUT BNC 24 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company e Measure the magnitude of the initial voltage jump on the oscilloscope Since the VOLTAGE 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 r
37. lium 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 800 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 5 agar with 1M KCl w v While still hot the agar can be drawn into polyethylene tubing using a syringe or vacuum line S
38. logy In the equivalent circuit shown to the right the cells of the epithelium are represented by a resistor Reo in series with a voltage source E 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 since the tissue is bathed by symmetric solutions For simplicity we have left out a series solution resistor Since parallel conductors add the transepithelial conductance Gi the conductance between M the mucosal solution and S the serosal solution is given by G G G Since resistance is the inverse of conductance Gi 1 R then the transepithelial resistance is given by _ RR R R t and from Ohm s law the current flow i around this circuit is E E R R Again using Ohm s law we find that the voltage difference across the epithelium Vm Vs V is equal to the current flow through the tight junction E R NERT ER c j 20 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company or through the cellular pathway R ER ER V E iR E 0 C a CU c f R R R R R Inspection of the above equations reveals three interesting features e The magnitude of the transep
39. mV uyA This input is inactive when GATE INPUT is high EXTERNAL TIMER IN Activated by selecting Ext Timerin the MODE control block A TTL signal at this BNC switches the amplifier between voltage clamp TTL low and current clamp TTL high modes Finally two LED s are provided to indicate overload conditions in the clamp amplifier or input amplifier circuitry EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Commands The VOLTAGE control comprised of a 10 turn potentiometer and a three position toggle switch adjusts the holding potential effective in voltage clamp mode The operational range is adjustable from zero to 100 mV with the polarity selected by the toggle switch The current control is also comprised of a 10 turn potentiometer and see a three position toggle switch This control adjusts the holding current VOLTAGE CURRENT effective in current clamp mode and the operational range is ak adjustable from zero to 1 mA with the polarity selected by toggle E switch Meter The METER section is comprised of a 3 5 digit LCD a 2 WARNER VOU r INSTRUMENT position toggle switch and the power switch for the unit A CORP The METER toggle switch MODE selects between current uA rowna and voltage mV readings Full scale is 1 900 uA and 199 dis i DAR mV for current and voltage positions respectively Either EC 800 Epithelial Voltage Clamp current or voltage can be
40. on e g 1 2 of Rmeas for low resistance epithelia it can be 50 or greater of Rmeas An alternative approach for measuring Rs 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 tissue 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 800 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 19 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company 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 morpho
41. rea In this section we briefly overview how the fluid resistance can be rapidly estimate 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 capacitor 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 YRR l e R l e t22 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 RiC 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 sing
42. 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 800 uses a 110 V current passing amplifier making this an instrument of choice for leaky epithelia The amplifier also has outputs which can be easily connected to chart recorders oscilloscopes or computers Minimum equipment requirements are i Acurrent voltage clamp amplifier This is essential to record the transepithelial voltage the transepithelial resistance and the short circuit current Isc 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 resistance ii A computer with acquisition har
43. sosesosssosssscsssessese 9 Connecting to dine DOWER rroi ee ORE NR aE EE A EE te A O E R oct 9 High voltag utputss ete eee tg ttem te oet etg eet aah eeu e e oe etg eee te e et uere 9 He dstagesa ett t e tr REN EO n tt ree etg eet ee oret ree rne Do e n e UR eae 9 Connecting the headstage eese esee eene enet tense tenen testes tense en testen EEE 10 SETUP AND INITIAL TEST eco eren eene et Treo So Enna eoe eU aen no bere ar pu etae tontos ea ena ssoneseonsseseven 10 loire Ep 10 Ghaibers 2 iiit HO e He e a de ee Hl e RETE I Hoe e drerit 10 JUDA 12 El CtFOllCsua iaa e e eie d eec et iu tbe d ert ect etc SP erbe ende ee BS 13 uarie 14 InitialcontroLsettingss eR uet eese Minis tuse redhead epe ce ted perle Mice 14 Monitoring model membrane voltage eese eene entente tenter nennen nente nene 14 Checking zero voltage clamp eese eee ELEN E tenen nentes sten seitens enne 14 Check voltage clamp command cccccccccesseceesseeeseceenseceeneecuseeesaeceeaaeceeseecsaeeeesaecescecseaeeceeeeeaeceeaaecenees 14 Checking clamp current on panel meter eese eene eene tenente enne nentes entente nnne 15 Fluid Resistance compensation eese esee eite entente AAEE nest e AS enne RE ENNEA IREE 15 COOPERATION E EE CS WT AR RU ER 16 Connecting the EH C 800 sciecscisecisecccicescessnsssencsdecsesesndssessestscenenasssunedsduecscnasseenerseunedsuassesnsceses UN nra pere
44. t 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 C C C Cy Cit C C R R _ R Ry R C R C R C R4C 1 1 R R R Ry RC R C RR C RR Cy 1 R R R R C R C 7 RjR C R C l 25 EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Note that a five parameter model for simplicity we ignore the solution resistance Rs 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 us
45. ter with push button convenience Y Clamp Speed Selection up to 10 ps Three clamp speeds provide optimum 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 The model membrane circuit simulates a preparation to provide convenient operational checks of the clamp Internal circuits are protected against the invasion of corrosive saline solutions by a watertight seal Y Choice of Compliance Voltage The high voltage compliance of the EC 800 120 V is important for studies of low resistance leaky epithelial cells and in applications in which long agar leads in the current passing circuit produce large voltage drops which must be compensated Additionally high compliance aids in charging large membrane capacitances typical of epithelial tissues resulting in faster settling times and improved overall clamp performance Studies with small tissue samples or monolayers in set ups with low access resistance may not require
46. to select for zero current clamp or for zero voltage clamp for current clamp mode or voltage clamp mode respectively All clamp commands current voltage and external are deactivated with a TTL high signal applied to this BNC Grounds Both circuit black and chassis green grounds are provided at rear binding posts The instrument is shipped with the two grounds connected via a shorting bar If needed this shorting bar can be disconnected allowing separation of the grounds Separating these grounds can in some cases result in a lowering of the 60 Hz noise level however experimentation will determine which is best for each set up Additional components and comments Connecting to line power The model EC 800 is supplied with a 3 conductor line cord One conductor provides a connection between the instrument housing and the power system 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 High voltage outputs Headstage The headstage has inputs for the 11 V1 V2 and 12 electrode cables and a three position switch selecting on off or test modes In addition test mode has a two position toggle switch selecting for Vmem or Fluid Resistance Moreover the headstage
47. urning the ten turn dial until zero is read on the digital meter The fluid resistance is read from the dial 0 100 Q outputs to data visualization and storage devices and are described below V Monitor x10 Output is the differential voltage V1 V2 multiplied by 10 I Monitor 10mV uA Output voltage is proportional to the current flowing through the preparation Mode This control block represents the main control section on the front panel and is CLAMP RESPONSE V CLAMP o comprised of a five position MODE r pes aMPuFY SELECTOR switch various LED indicator J E Q i MEMBRANE lights the CLAMP RESPONSE push button R MEMBRANE RANGE B cr a i eo ko the R MEMBRANE RANGE push button and EXT SIGNAL M usn r INPUT Cc two INPUTS BNC s Ve 100mVv V EXTERNAL INPUT CLAMP Tor TRR The MODE SELECTOR SWITCH is used to AMPLIFIER AMPLIFIER O QO select among the various operational e Q QL OVERLOAD modes available in the instrument EC 800 EC 800LV Manual Rev 050815 WARNER INSTRUMENTS A Harvard Apparatus Company Available selections are V Clamp Amplify C Clamp R Membrane and Ext Timer LED s indicate the MODE selection The meaning of MODE selections are shown in the table below 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 HEAD
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