HiClamp Manual
Information in this document is subject to change without notice. No part of this document may be reproduced or transmitted without the express written permission of Multi Channel Systems MCS GmbH. While every precaution has been taken in the preparation of this document, the publisher and the author assume no responsibility for errors or omissions, or for damages resulting from the use of information contained in this document or from the use of programs and source code that may accompany it.
Table of Contents 1 1.1 Introduction About this Manual 1 1 2 2.1 2.2 2.3 Important Information and Instructions Guarantee and Liability Operator's Obligations Important Safety Advice 2.3.1 High Voltage 2.3.2 Requirements for the installation 2.3.3 Handling of the motor driven axes 2.3.4 Handling of the chlorided silver wires 2.3.5 Handling of recording electrode needles 3 3 3 4 4 4 5 5 5 3 3.1 3.2 3.3 3.
HiClamp Manual 6 6.1 6.2 Data Analysis Data File Organization DataMining 6.2.1 Measuring the current amplitudes 6.2.2 The e-Labbook of the HiClamp DataMerger 6.3.1 Statistic 6.3.2 Example of a curve fitting 6.3.3 Example of curve fitting using “Global Fit” 39 39 40 43 45 46 47 47 52 7.2 Examples The voltage activation curve of Kv1.4 Current-Voltage activation curve for KV1.4 The voltage activation curve of NaV 1.5 55 55 56 58 8 8.1 Troubleshooting Troubleshooting 61 61 9 9.
1 Introduction 1.1 About this Manual This manual comprises all important information about the first installation of the HiClamp hardware and software and about the daily work with the HiClamp. It is assumed that you already have a basic understanding of technical and software terms. Thus, no special skills are required to read this manual. If you are using the HiClamp for the first time, please read the "Important Safety Advice" before installing the hardware and software.
2 Important Information and Instructions 2.1 Guarantee and Liability The general conditions of sale and delivery of Multi Channel Systems MCS GmbH always apply. The operator will receive these no later than on conclusion of the contract. Multi Channel Systems MCS GmbH makes no guarantee as to the accuracy of any and all tests and data generated by the use of the device or the software. It is up to the users to use good laboratory practice to establish the validity of their findings.
HiClamp Manual 2.3 Important Safety Advice Warning: Make sure to read the following advice prior to installations of the HiClamp. If you do not fulfill all requirements stated below, this may lead to malfunctions, breakage, or even fatal injuries. Obey always the rules of local regulations and laws. Only qualified personnel should be allowed to perform laboratory work. Work according to good laboratory practice to obtain best results and to minimize risks.
Important Information and Instructions 2.3.3 Handling of the motor driven axes Do not move the axes by hand. Breakage may occur. Always use the software controls to move in vertical or horizontal level. The capillaries of the probe are sharp and may lead to injuries. Stay at a safe distance during operation and protect your eyes. Especially take care not to move your hands in the range of the vertical axis. Do not try to plug anything other than 0.
3 HiClamp 3.1 Welcome to the HiClamp The HiClamp is a fully-automated all-in-one solution for high-throughput functional secondary screening of drug targets based on the standard Xenopus expression system. The HiClamp allows an automated recording using the TEVC method unattended and over night. Main features Compact and functional design, works fully computer-controlled. High performance TEVC amplifier, records current of up to 105 μA. Export features for further data evaluation.
HiClamp Manual The program sequence is an ensemble of operations that are executed sequentially according to a set of icons that are stored in the program sequence. Then, each icon corresponds to a given procedure. For example, the oocyte icon will contain the type of message injected into the oocytes, the number of cells available etc.
HiClamp 3.2 Hardware The HiClamp hardware The HiClamp is compatible with standard lab equipment and can be easily integrated in your working environment. Software controls replace any knobs on the device. The HiClamp is straightforward and easy to operate; handling does not require special skills or special equipment. Recording and cultivation of Xenopus oocytes is performed using disposable standard 96 well plates, which are commercially available from several providers.
HiClamp Manual 3.3 Setting up the HiClamp Setting Up the HiClamp 1. Provide a power supply in the immediate vicinity of the installation site.
HiClamp Connecting the HiClamp to the provided computer (USB) Connect the HiClamp directly to the provided computer by the provided USB cable. The appropriate connector on the rear panel of the computer is tagged with a "HiClamp" label. Note: Please do not use one of the USB 3.0 ports of your computer for connecting the HiClamp. Power In for the HiClamp (24 VDC HiClamp) Connect the HiClamp to the power supply unit here. This power supply powers the HiClamp main unit only.
HiClamp Manual 3.4 Mechanical Functionality of the HiClamp The specific characteristic of the mechanical components of the HiClamp device is their mobility in three dimensions. The movement of the table complex in dependency of the movement and adjustment of the measuring complex is the main idea of the HiClamp’s functionality. The table complex moves on track rails in X, Y and Z direction under software control of very precise motors.
HiClamp For washing the cell between two measuring cycles there is a washing station placed between the well plates. The well plate on the right hand side is called “Feeder Plate” and contains the oocytes to be tested. The well plate on the left hand side is called “Compound Plate” and contains the solutions of interest. Each compound well should be filled with 230 μl. Additionally a micro stirrer is placed inside which is moved by magnetic forces.
HiClamp Manual Automated Loading of Oocytes from Compound Well Plate The oocytes are stored in the 96 well plate called feeder plate on the right side of the table. They will be automatically transferred to the compound plate on the left side for measuring the cells. For transportation and measuring the oocytes are carried in the basket.
HiClamp Cartoon of the automated oocyte transport from 96-well feeder plate to the oocyte basket: The oocyte is in the selected well of the feeder plate (1). The suction tube is lowered into the well (2). The oocyte is aspirated (3). The suction tube moves to the basket (4). The suction tube is lowered directly above the basket (5). The suction tube emits the oocyte (6). The oocyte is placed in the basket (7). The basket moves with the oocyte beneath the position of the TEVC electrodes (8).
4 Software 4.1 Program Concept To maximize the system capacity while simplifying the use of the HiClamp device for data acquisition and analysis, HiClamp and the corresponding data analysis program are built around an object oriented concept. In such concepts the object, this is the icon, contains information to effectuate the appropriated and desired operation. In HiClamp, objects are represented as icons that symbolize the action to be executed.
HiClamp Manual Caution: You have acquired a high performance data acquisition and analysis computer. Do not modify the system, do not install new hard- or software, or another operating system without asking MCS or your local retailer for advice. MCS recommends not to install virus scanners or firewalls because these programs are known to interfere with the HiClamp to computer connection and with the data transfer.
5 TEVC Recording Protocol 5.1 Preparations for Recording Installing solutions Solutions applied to the washing station are pumped by the peristaltic pumps. Verify that the tubings are properly disposed and placed in the solution. Clicking the “Manual” icon activates the manual command dialog which allows the manual control of the pumps. Filling of all used tubings is a precondition for proper function of the washing station during recording.
HiClamp Manual 5.2 Electrode Alignment Mounting and adjusting the electrodes First, mount the provided glass capillaries on the measuring head. Use a binocular and adjust the tip distance between 300 and 400 μm. Also be careful to align the tips to be at about the same height. Then, fill the capillaries with either 3 M KCl or with a mixture from KCl and KAc which reduces crystallization (1 M and 1.5 M, respectively). Filling should not exceed about 70 % of the capillary length.
TEVC Recording Protocol Press on the Electrodes icon to open the Electrodes window. Press on the "Down" button for slowly moving the electrodes down towards the oocyte basket. Use the camera for optical control. Lower the electrodes progressively into the center of the basket, just a little bit deeper than necessary to penetrate the oocyte. Note that the camera is mounted on a rotating arm to facilitate the adjustments.
HiClamp Manual 5.3 Test Model Cell 5.3.1 Testing the Amplifier with the Test Model Cell Before you start to do real experiments with oocytes, we recommend to get familiar with the HiClamp using the Test Model Cell (TMC). The TMC mimics the electrical (passive) properties of a real oocyte impaled with microelectrodes (electrode resistance ~450 kOhm, membrane resistance 100 kOhm, membrane capacitance ~ 200 nF)).
TEVC Recording Protocol 5.3.2 Tuning the Amplifier The build in digital amplifier can perform an automatic capacitance compensation in real-time during the voltage-clamp recordings. This is an unique feature which is necessary to maintain stable clamping during the movements of the oocyte basket. This automatic capacitance compensation can be switched on and off with the "Auto On" check box. Please note that "Auto On" should be checked during all recordings.
HiClamp Manual Now, after tuning, you can test the performance of your system with a simple “"Voltage Step Protocol" (IV protocol) with, for example, 10 short voltage steps from -120 to +60 mV in 20 mV increments.
TEVC Recording Protocol After executing and completing this voltage step protocol, the data display should show current traces looking like this. Please see the picture below. The traces shown above have been recorded with gain 1000 / 100 and an 1 kHz recording filter.
HiClamp Manual 5.4 Selecting Oocytes for TEVC Recording Selecting Oocytes Please read chapter "Preparation of Oocytes" in Service and Maintenance. Plating Oocytes Please read chapter "Plating Oocytes" in Service and Maintenance. Loading the oocytes plate Place the 96 microtiter plate containing the oocytes in the right slot on the table. Tighten the fixation of the plate (lower right) and verify that the holding screw is properly secured.
TEVC Recording Protocol Selections are done by a double click on the icon in the program sequence. A double click on the first oocyte icon opens the “Load Plate” dialog. This window allows to enter parameters about the well plate and about the type of DNA injected into the oocytes as well as the position of this oocytes etc. A double click on the second icon opens the “Test Well” dialog.
HiClamp Manual If you turn on the data acquisition by checking the box "Acquisition", the second icon will change it´s appearance and "A/D" will be displayed instead of “W”. If you click the button "Voltage Command" the respective dialog opens which allows you to define voltage protocols in step and ramp mode. Pushing the button Play will start HiClamp. The button “Play” will be replaced by Stop as soon as the sequence is activated. Hitting the Stop button will stop the procedure.
TEVC Recording Protocol 5.6 Icons and the associated Effects Oocyte Loading This icon is used to select the oocytes that will be taken for the measurements. So it is normally the first of a program sequence. Oocytes setting, such as the injected message, number of cells, date of injection etc. are stored in the corresponding data file. Access to these parameters is done by a double click on the "Oocyte Loading" icon and filling the appropriate parameters.
HiClamp Manual After clicking on "Done" the list window appears. Once defined you can change the active definition with the "Edit" function. Wash/Test The "Wash/Test" icon is used to move the oocyte into the wash station where compounds can be washed or a test solution can applied to test the oocyte response. Parameters corresponding to this step can be accessed after a double click on the icon.
TEVC Recording Protocol Following a double click on the desired icon the condition window is displayed. “Go To Step” indicates the icon to which the “if” is returning when the condition are not met. The “Leak” (expressed in μA) and current (expressed in μA) are used for the evaluation of the “if”. Unless the cursor are set up (see below) values are determined on the 10 first data points for the leak current and on the entire data epoch for the current.
HiClamp Manual Loop no increment The loop with no increment acts almost similarly to the loop. However, at difference with the loop with increment, this loop causes no modification of the compound to be measured. This loop can be used to measure a multiple number of time the same compound. Another important difference between loop with increment and without increment is observed in the loop associated window.
TEVC Recording Protocol Loop including a variable component The loop including a variable element can be used for defining voltage step protocols. Whenever you include the place holder "x" instead of a number, for example in the "Voltage Command" window and you loop back to this definition, you can define in the appearing "LoopX" window the values replacing the "x" incrementing with every loop count. In the shown example the potential steps go from -100 mV to +20 mV in 20 mV increments.
HiClamp Manual 5.7 Data Acquisition Icons Execution of the icons described above allows the control of HiClamp movement and action. Additional icons are, however, necessary to perform data acquisition. Three icons control the data acquisition and can be used in the program sequence. At the end of each data acquisition the window DataPlot is turned on to display the last recorded data period.
TEVC Recording Protocol The Notebook An electronic laboratory notebook is built in the HiClamp program. Access to this logbook is automatic when the first sequence is activated. The notebook window cannot be turned off during the experiment but can be minimized and recalled by a single click on the corresponding icon. The notebook window is divided in two parts. The upper section presents the notes that have been entered during the experiment. These notes are stored in the corresponding *.
HiClamp Manual 5.7.2 The concentration activation curve A first example of a program sequence is the determination of the concentration activation curve for a ligand gated channel. To establish such relationship the response of a cell exposed to a series of ligand concentration is measured and the concentration activation curve is obtained by plotting the peak of the evoked current (or, in some cases, the area under the curve) as a function of the logarithm of the agonist concentration.
TEVC Recording Protocol In the first step the oocyte is loaded, as previously described. The voltage protocol is defined in the voltage command window that can be seen after opening the control of the step 2. Opening the voltage command, by pressing the corresponding button, reveals the control variables accessible for the definition of single or multiple voltage steps.
HiClamp Manual A single recording data epoch (or sweep) is defined by its holding voltage -100 mV, and the number of steps the values of which are indicated in "LoopX" window. The values above indicate which steps have to be sequentially performed. For example, to record the activity of a potassium channel it would be necessary to step the voltage between different values ranging from -50 to + 10 mV starting from a holding potential of -100 mV.
6 Data Analysis As the HiClamp is intended to perform experiments in an unattended manner this means that data captured by the robot should be self containing and easily identifiable. Typical experiments will run for hours and often during day or night. Moreover, data concerning a particular set of experiments may be captured in a single day or on several days and the data analysis software must allow flexible analysis to be performed and regrouping of data coming from different experiments.
HiClamp Manual 6.2 DataMining The DataMining was specially designed for the treatment of data captured with the HiClamp. The aim of this program is to provide the user with a convenient interface allowing efficient data retrieval and analysis. Upon activation of this program a window resembling the HiClamp environment is displayed. The screenshot shows the main window of DataMining software.
Troubleshooting The program sequence that is displayed in the middle panel is identical to the program sequence used for the control of HiClamp. The lower panel illustrates the sequence of events that took place during the data acquisition in a “chart paper like” recording. In this example you can see that the HiClamp took three oocytes from the loading plate before finding one responsive cell as indicated by the three oocyte loading icons that are seen in the initial section of the sequence.
HiClamp Manual A single click on the lower left corner of any of these small graphs displays the corresponding current traces as shown in the screenshot. Another form of representation for the same data can be obtained by selecting the mode Plot Seq. When this mode is selected data are represented as on a chart paper (see screenshot).
Troubleshooting 6.2.1 Measuring the current amplitudes Measurement of the peak ACh-evoked current is carried out automatically within boundaries defined in the Set Cursor command. This command is activated by a single click on the button presented in the right panel. Activation of the Set Cursor changes the display as follows. Several cursor options are possible allowing measurements at the cyan and blue lines.
HiClamp Manual Once cursors are set the ensemble of the data captured in a single run can be analyzed by choosing the calculator mode and by a single click on the A/D after activation of the calculator that is display in the right part of the commands. Following the selection of the A/D the calculator should display the number 7, which corresponds to the position of the A/D in the data acquisition program.
Troubleshooting 6.2.2 The e-Labbook of the HiClamp The electronic laboratory book that opens when HiClamp is set to run is displayed when the sequence is read in DataMining. In this example the e-labbook is presented as follows: Note that each sequence is indicated with the date, time of activation and end. A double click on a selected sequence automatically opens this sequence in DataMining.
HiClamp Manual 6.3 DataMerger DataMerger program was especially conceived for the analysis of data selected with DataMining and for combining experiments acquired in different session. Typically, experiments obtained on day one might need to be compared with experiments from day two or analyzed simultaneously. DataMerger allows analysis of single point measurements, multiple measurements, concentration activation or inhibition curves, or voltage-dependent channel activity.
Troubleshooting 6.3.1 Statistic Activation of the statistic button displays the average and SEM for the points that can be fitted by a single or dual Hill equation. Note that each curve can be fitted separately and the mean of curve can later be computed and analyzed. Values corresponding to the original data points and normalized values are stored in an excel compatible format by activation of the excel icon.
HiClamp Manual When a curve is selected the corresponding data points are represented as stars and the curve fitting parameters are displayed in the right window. To optimize curve fitting it is often recommended to provide a first guess of the parameters. In this example we shall select 100 M for the EC50.
Troubleshooting A continuous line corresponding to the guess parameters is displayed upon keying the desired value and the display should appear as shown. Pressing Fit Selected Data initiates the curve fitting procedure and result with the best fit is displayed.
HiClamp Manual Values indicated in the windows correspond to the best fit obtained from the guess starting point. Pressing Fit All Data initiates the curve fitting procedure on each of the curve and results in: Pressing the Statistics button computes the standard error of mean and display the average points together with the SEM.
Troubleshooting Selecting the average curve activates the display of an additional button and computation of the Mean of Curves can be effectuated. As in this example the mean of curves fits nicely the average curve no further adjustment is necessary. Data can now be removed by selecting Delete Data in the menu. Leaving only the average curve in the display. Data can be further processed and saved as an excel data sheet for easy reporting by pressing the Excel button.
HiClamp Manual The corresponding Excel data sheet should be as following: 6.3.3 Example of curve fitting using “Global Fit” An alternative to SIMPLEX curve fitting is to fit all the available data using the technique of Global Fit. To activate the global fitting procedure it is first necessary to select the option in the Computation menu bar.
Troubleshooting Once the selection of Global Fit is made a new option set is displayed allowing the selection of single or dual Hill equation. In our example data points are readily fitted by a single Hill equation and activation of the Global Fit yields the following results. Result from the Global Fit are indicated in the new floating window and best fit obtained with this method is illustrated by the continuous line. Note the similitude of results obtained with the SIMPLEX curve fitting method.
HiClamp Manual Open DataMerger program. Open the different *.mat produced with DataMining. Data will appear in DataMerger and sorted according to the *.mat name. To merge data press CTRL and click on the first data set. The pointer will be changed from the arrow to a circle indicating that the merge is activated. Maintaining the CTRL key, click on the panel you want to merge. The two panels should change and results should be merged as follows: Using the Option “Plot all” displays all the curve at once.
Troubleshooting 7 Examples 7.1 The voltage activation curve of Kv1.4 Two alternatives can be used for the measurement of the voltage activation curve by measuring with the current activated during a voltage step and measuring of the relaxation current. The program sequence for both example was the same and voltage steps were defined by setting Step 1 to -50, 20 increments of 5 mV each. Setting Step 2 to -50 mV evoked a tail current of sufficient amplitude for the measurement of the relaxation.
HiClamp Manual Placing the crosshair (in the crosshair mode) immediately at the end of the capacitance discharge and at the end of the voltage step allows a first estimation of the current activated by the voltage step. Ideally, measurement of the current should be subtracted from the current passing through the passive properties of the membrane which is usually obtained by subtracting the current measured for the same voltage step in presence of a selective blocker. Positioning the crosshair for KV1.
Troubleshooting Data can be fitted using the typical procedure of selecting the desired curve, selecting the initial parameters, and activation of the curve fitting procedure. Data are fitted using a single Boltzman equation. Note that results of the curve fitting are applied to the IV curve allowing to compare the result of the fit with the data. Plot of the conductance for KV1.
HiClamp Manual Voltage activation measured on the relaxation of tail current 7.2 The voltage activation curve of NaV 1.5 To determine the voltage activation relationship for NaV 1.5 a standard series of voltage steps are applied at periodic intervals. Voltage steps of regular increments can be set in the voltage command interface either from Wash/Test, A/D or Plate with acquisition. Currents recorded in response to a series of voltage steps in an oocyte expressing the NaV 1.
Troubleshooting Plot of IV curves for the NaV 1.5 The plot of the conductance as a function the voltage step is readily obtained by activating the conductance button. Note that the reversal potential is automatically proposed in the Erev window, please see the next screenshots. Conductance plot and reversal potential Adjustment of the reversal potential deduced from the left screenshot result the graph presented in the right screenshot.
HiClamp Manual Curve fitting of the desired curve is done by selecting the curve of interest and with the Boltzman equation. Following selection of the curve, done by a single click on any one of the corresponding symbol, a window displaying the parameters of the Boltzman equation is displayed.
Troubleshooting 8 Troubleshooting 8.1 Troubleshooting Most problems occur seldom and only under specific circumstances. In most cases, it is only a minor problem that can be easily avoided or solved. If the problem persists, please contact your local retailer. The highly qualified staff will be glad to help you. Please inform your local retailer as well, if other problems that are not mentioned in this documentation occur, even if you have solved the problem on your own.
9 Service and Maintenance 9.1 Service and Maintenance Please make sure that you read this chapter thoroughly. Proper handling and cleaning of the HiClamp will ensure reliable and long life time of the equipment. Most often, troubles are simply due to mishandling. See also chapter "Troubleshooting". 9.1.1 Cleaning of the Basket The basket is the most delicate part of HiClamp.
HiClamp Manual 6. Select the option Adjust Basket from the menu bar. 7. Select the basket position to Load Basket. The basket will move into the loading position and the suction tube is lowered towards the basket. The suction tube will be used as the reference point to which the basket needs to be aligned. 8. Use the two small Allen keys screws to bring the basket in the closest juxtaposition with the suction tube but without contact. 9.
Service and Maintenance 9.2 Preparation of Oocytes Rough selection by filtration 1. Fill a 100 ml beaker with 80 ml of Barth's solution. Place the oocyte mesh sieve into the beaker for grading the oocytes by size. The mesh of the sieve should be completely immersed in the fluid. 2. Pipette an amount of oocytes onto the sieve. Approximately half the sieve should be covered with oocytes. Too many oocytes on the filter will lead to an inefficient filtration. 3.
HiClamp Manual 9.3 Plating Oocytes You need a well plate filled with Barth's + gentamicin ???? Important: Visually check all well plates before use. Use only unsterile and very flat plates. Sterilization may lead to warped plates. Warped plates will decrease the injection and expression efficiency. 1. Aspirate a number of oocytes from the dish with a transfer pipette by applying a low negative pressure, for example, with a rubber ball. 2.
10 TEVC Recording Background 10.1 TEVC Recording Background About TEVC Recording with the HiClamp The small-sized digital TEVC amplifier is integrated within the HiClamp and fully computercontrolled. It operates either in true current clamp or voltage clamp mode and uses a PI (Proportional Integral) based technique. The ClampAmpC records up to ± 105 μA of current, and the 16-bit A/D-conversion together with the over-sampling method results in an overall resolution of 1 nA.
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Appendix 11 Appendix 11.1 Technical Support Please read the "Troubleshooting" part of the manual or help first. Most problems are caused by minor handling errors. Contact your local retailer immediately if the cause of the trouble remains unclear. Please understand that information on your hardware and software configuration is necessary to analyze and finally solve the problem you encounter.
HiClamp Manual 11.
Appendix 11.3 About Preparation of Xenopus Oocytes The materials and methods described in the following are kindly provided by the Bayer AG, Leverkusen. The described procedures have been optimized over several years for best performance of the oocytes and highest throughput. We recommend that you follow the instructions for obtaining best results. Refer to standard protocols if you need more detailed information on the subject.
HiClamp Manual 11.3.2 Chemicals Collagenase For defolliculation: Fresh 1.52 mg/ml collagenase in Barth’s solution without Calcium. The concentration has to be optimized according to the collagenase batch and experimental conditions, see also chapter "Defolliculation". Do not prepare solutions in advance as collagenase activity may decrease rapidly even if the solution is stored at -20 °C. Collagenase from Cl. histolyticum ca. 0.17 to 0.28 U/mg (according to Wünsch,1963) lyophilized.
Appendix 11.3.4 Collagenase Digestion and Defolliculation Ovarian tissue contains immature and mature oocytes, as well as connective tissue from which the oocytes must be separated. Ovarian tissue should be removed completely by collagenase digestion. Oocytes are enveloped in a follicle cell layer which can cause trouble when plating oocytes into well plates. Remaining pieces of follicular tissue causes oocytes to stick to the walls.
HiClamp Manual Fine selection Use a stereo microscope and the golf-club shaped glass tool to check each single oocyte for the following criteria. Outer form: No visible damage of the cell. Well separated colors (dark and light brown). No residues of follicular tissue. Size: Uniform size of about 1.2 mm. Note: Selecting oocytes is an important step. Perform it very carefully to obtain best results. 11.3.6 Plating Oocytes You need a well plate filled with Barth's + gentamicin.
Appendix 11.4 Contact Information Local retailer Please see the list of official MCS distributors on the MCS web site. User forum The Multi Channel Systems User Forum provides the opportunity for you to exchange your experience or thoughts with other users worldwide. Mailing list If you have subscribed to the Mailing List, you will be automatically informed about new software releases, upcoming events, and other news on the product line.
