Multichannel Systems HiClamp User Manual
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. In no event shall the publisher and the author be liable for any loss of profit or any
other commercial damage caused or alleged to have been caused directly or indirectly by this
document.
© 2012 Multi Channel Systems MCS GmbH. All rights reserved.
Printed: 16. Mai 2012
Multi Channel Systems MCS GmbH
Aspenhaustraße 21
72770 Reutlingen
Germany
Fon +49-71 21-90 92 5 - 0
Fax +49-71 21-90 92 5 -11
info@multichannelsystems.com
www.multichannelsystems.com
Microsoft and Windows are registered trademarks of Microsoft Corporation. Products that are
referred to in this document may be either trademarks and/or registered trademarks of their
respective holders and should be noted as such. The publisher and the author make no claim
to these trademark.
Table of Contents
1 Introduction 1
1.1 About this Manual 1
2 Important Information and Instructions 3
2.1 Guarantee and Liability 3
2.2 Operator's Obligations 3
2.3 Important Safety Advice 4
2.3.1 High Voltage 4
2.3.2 Requirements for the installation 4
2.3.3 Handling of the motor driven axes 5
2.3.4 Handling of the chlorided silver wires 5
2.3.5 Handling of recording electrode needles 5
3 HiClamp 7
3.1 Welcome to the HiClamp 7
3.2 Hardware 9
3.3 Setting up the HiClamp 10
3.4 Mechanical Functionality of the HiClamp 12
4 Software 17
4.1 Program Concept 17
4.2 Installing the HiClamp Software 17
4.3 Starting the HiClamp 18
5 TEVC Recording Protocol 19
5.1 Preparations for Recording 19
5.2 Electrode Alignment 20
5.3 Test Model Cell 22
5.3.1 Testing the Amplifier with the Test Model Cell 22
5.3.2 Tuning the Amplifier 23
5.4 Selecting Oocytes for TEVC Recording 26
5.5 Starting a Recording Sequence 26
5.5.1 A first and simple example 26
5.6 Icons and the associated Effects 29
5.7 Data Acquisition Icons 34
5.7.1 Measuring multiple oocytes 35
5.7.2 The concentration activation curve 36
5.7.3 Recording Voltage-Gated Channel activity 36
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Data Analysis 39
6.1 Data File Organization 39
6.2 DataMining 40
6.3 DataMerger 46
7 Examples 55
7.1 The voltage activation curve of Kv1.4 55
7.2 The voltage activation curve of NaV 1.5 58
6.2.1 Measuring the current amplitudes 43
6.2.2 The e-Labbook of the HiClamp 45
6.3.1 Statistic 47
6.3.2 Example of a curve fitting 47
6.3.3 Example of curve fitting using “Global Fit” 52
Current-Voltage activation curve for KV1.4 56
8 Troubleshooting 61
8.1 Troubleshooting 61
9 Service and Maintenance 63
9.1 Service and Maintenance 63
9.1.1 Cleaning of the Basket 63
9.1.2 Replacing or Realigning 63
9.2 Preparation of Oocytes 65
9.3 Plating Oocytes 66
10 TEVC Recording Background 67
10.1 TEVC Recording Background 67
11 Appendix 69
11.1 Technical Support 69
11.2 Technical Specifications 70
11.3 About Preparation of Xenopus Oocytes 71
11.3.1 Materials 71
11.3.2 Chemicals 72
11.3.3 Oocyte Removal 72
11.3.4 Collagenase Digestion and Defolliculation 73
11.3.5 Selecting Good Oocytes 73
11.3.6 Plating Oocytes 74
11.4 Contact Information 75
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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. Please see chapter "First Use of the HiClamp",
where you will find important information about the installation of hardware and software.
The printed manual and online help are basically the same, so it is up to you which one you
will use. The help offers you the advantage of scrolling through the text in a non-linear fashion,
picking up all information you need, especially if you use the index and the search function.
If you are going to read larger text passages, however, you may prefer the printed manual.
The device and the software are part of an ongoing developmental process. Please understand
that the provided documentation is not always up to date. The latest information can be found
in the HiClamp help. Check also the Multi Channel Systems MCS GmbH web site
(www.multichannelsystems.com) for downloading up-to-date manuals.
1
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.
Guarantee and liability claims in the event of injury or material damage are excluded when
they are the result of one of the following.
Improper use of the device.
Improper installation, commissioning, operation or maintenance of the device.
Operating the device when the safety and protective devices are defective and/or inoperable.
Non-observance of the instructions in the manual with regard to transport, storage, installation,
commissioning, operation or maintenance of the device.
Unauthorized structural alterations to the device.
Unauthorized modifications to the system settings.
Inadequate monitoring of device components subject to wear.
Improperly executed and unauthorized repairs.
Unauthorized opening of the device or its components.
Catastrophic events due to the effect of foreign bodies or acts of God.
2.2 Operator's Obligations
The operator is obliged to allow only persons to work on the device, who
are familiar with the safety at work and accident prevention regulations and have been
instructed how to use the device;
are professionally qualified or have specialist knowledge and training and have received
instruction in the use of the device;
have read and understood the chapter on safety and the warning instructions in this manual
and confirmed this with their signature.
It must be monitored at regular intervals that the operating personnel are working safely.
Personnel still undergoing training may only work on the device under the supervision
of an experienced person.
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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.
The product has been built to the state of the art and in accordance with recognized safety
engineering rules. The device may only
be used for its intended purpose;
be used when in a perfect condition.
Improper use could lead to serious, even fatal injuries to the user or third parties and damage
to the device itself or other material damage.
Malfunctions which could impair safety should be rectified immediately.
2.3.1 High Voltage
Electrical cords must be properly laid and installed. The length and quality of the cords must
be in accordance with local provisions.
Only qualified technicians may work on the electrical system. It is essential that the accident
prevention regulations and those of the employers' liability associations are observed.
Each time before starting up, make sure that the mains supply agrees with the specifications
of the product.
Check the power cord for damage each time the site is changed. Damaged power cords should
be replaced immediately and may never be reused.
Check the leads for damage. Damaged leads should be replaced immediately and may never
be reused.
Liquids may cause short circuits or other damage. Keep the power supply and the power cords
always dry. Do not handle it with wet hands.
2.3.2 Requirements for the installation
The HiClamp weighs more than 47 kg. Always grip it tightly and do not carry it alone,
but with the aid of another person.
The movements of the well plate carrier can lead to vibrations of the workbench on which
the HiClamp is set up. Therefore, the HiClamp must be set up on a rigid, vibration-free base.
The base must also be sufficiently solid to carry the weight of the device.
The HiClamp should be operated only in an air conditioned room. A room temperature
of 20 °C (or less) is recommended. Make sure that the device is not subject to direct sunlight.
It may overheat.
If the air cannot circulate freely around the external power supply, the device may overheat.
Do not shield the power supply by laying anything on top of the acryl glass cover.
The external power supply is only for use with the HiClamp. Do not connect it to any other
instrument.
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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 0.4 mm wire or the provided connectors into the
sockets of the recording axis. Damage may occur.
2.3.4 Handling of the chloride coated silver wires
The silver chloride coated silver wires are sensitive to light. Always keep them in dark during
storage. Make sure that all electrodes (including the reference electrodes) are still well-chlorided
before you start a recording. They should look dark grey, not shiny. The Ag/AgCl layer
deteriorates over time, leading to a DC offset and a voltage drift over time.
We recommend that you use the provided connectors to connect the electrodes to the vertical
axis. If you want to plug the silver wire directly into the sockets of the vertical axis, use only
0.4 mm silver wire for the electrodes. A wire with a greater diameter will damage the
connectors of the vertical axis irreversibly.
2.3.5 Handling of recording electrode needles
The needles are sharp and break easily. Always handle them with care.
In rare cases, the needles may splinter when pressure is applied. Stay at a safe distance
and protect your eyes.
Operation of the peristaltic pump
Empty the waste bottle at regular intervals, at least once a day if the HiClamp is in use.
The peristaltic pump must not be operated if the waste bottle is full.
Regular backups
You (or the administrator) should perform backups of the HiClamp data files at regular intervals
and to appropriate recording media for preventing data loss. Data loss may be caused by power
failure, system and software errors.
INI file modifications
If you remove or edit text of an INI file, the software may cause severe problems. Some INI files
relate to hardware functions. A modification of INI files may lead to malfunctions or even severe
damage of the hardware. Always keep a copy of the original INI file. Only advanced users should
modify program files. This warning message applies to all INI file modifications.
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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.
Integrated washing station with a peristaltic pump based four channel perfusion system.
Non-destructive usage of test compounds.
Small sample volumes (~200 μl).
Operation summary:
The HiClamp functionality is based on the use of a novel system in which the oocyte is exposed
to a test solution by moving it physically into the solution of interest, whereas in a standard
system the solution is applied on the cell. The test solutions are disposed in a 96 microtiter plate
which is fixed on a table (in the left part). The oocyte is fixed in the support held (oocyte basket)
in the center of the automate and the table moves to immerge the cell in the desired solution.
The table moves in X, Y and Z direction under software control of very precise motors.
The HiClamp software program is an “icon based” program and was designed to simplify the
user interface while maximizing the possibilities for electrophysiological recordings. No special
programming knowledge is required to use the HiClamp software.
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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. At difference, the icon corresponding to the amplifier
will store the information controlling the amplifier gain, filter conditions, voltage clamp set up,
voltage steps etc. To access the content of a given icon the user double clicks on the icon stored
in the program sequence.
After defining the recording protocol, simply start the recording sequence by mouse click.
The run will proceed automatically until finished or interrupted by the user.
The HiClamp software
Operate the HiClamp, collect and evaluate the data by using the HiClamp software. The easyto-use graphical user interface of the HiClamp software makes daily work with the HiClamp
quick and easy. Recording is started by a single mouse click. The HiClamp controls the run
for all 96 oocytes automatically. Thus the recording can go on overnight, unsupervised.
You can define different experimental setups not only for different well plates, but also
for specific selections of wells on the same plate. Several control features to save time and
compounds are provided.
Automated analysis features are included in the HiClamp software, but you can export the data
to your custom evaluation software as well. You can graph the data and generate reports with
the HiClamp software. The large amount of data generated by the HiClamp can be managed
with the included DataMerger software.
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3.2 Hardware
The HiClamp hardware
HiClamp
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. The oocytes are plated into the
wells in a couple of minutes and can be kept for several days. The oocytes do not have to leave
the plate anymore; you can easily transfer the oocytes from the incubator to the HiClamp and
back again.
A quick alignment process under camera control guarantees that the glass microelectrodes
in the measuring head impale the oocyte precisely centered before recording.
The ClampAmpC is a specifically designed digital TEVC amplifier and is completely automated.
The Syringe System arranges the aspiration of the cell when loading it into the basket via low
pressure.
The Perfusion System with inlet and outlet tubes and external peristaltic pump is for managing
the wash and test station placed between the 96 well plates on the table.
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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.
Important: Make sure that you do not place any AC voltage sources, that is, any electrical devices
or cables in the immediate vicinity of the HiClamp robot (especially not near the amplifier or near
any parts belonging to the perfusion system, for example, the bath reservoir or the waste bottle),
as they can introduce a 50 Hz noise or other electrical interferences to your recordings. Move
electrical devices or cables a few inches away if you observe any problems.
2. Place the HiClamp on a rigid, stable, and vibration-free base in an air conditioned room.
3. Place the computer and its accessories next to it. Make sure that you do not place the monitor
or the computer too near to the HiClamp robot or the perfusion system.
Connecting the HiClamp
Connect all cables as described below.
Warning: Carefully lay and secure the cords. Remember that someone could easily trip
over a loose cable.
Note: All electrical connections are clearly marked, and the plug coding prevents confusion.
The cords should be plugged in without the use of excessive force.
The following illustration shows the back panel of the HiClamp.
Connecting the video camera (Cam)
The USB cable of the video camera is fixed to the rear panel of the HiClamp.
Please connect the cable of the video camera to an USB port of the computer.
Connecting the peristaltic pump stand (Pump)
Plug the female connector into the male socket of the peristaltic pump. Then plug the male
valves connector into the female socket on the HiClamp's back panel.
On / Off Toggle Switch I / O (Power)
Toggle switch I / O for turning the HiClamp device on and off.
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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.
Power In for the Cooler (24 VDC Cooler)
Connect the Cooler to the power supply unit here. This power supply powers the Cooler main
unit only.
Warning: If the air cannot circulate freely around the external power supply, the device
may overheat. Do not shield the power supply by laying anything on top of it. Make sure
it is not exposed to direct sunlight.
Note: All electrical connections are clearly marked, and the plug coding prevents confusion.
The cords should be plugged in with no excessive force.
The Peristaltic Pump Perfusion System with four Channels
For washing the oocyte between two measuring circles there is a washing station placed
between the well plates. The washing station can also be used for applying test compounds
to the cells.
The test and washing station is connected to five tubes: Four inflow and one outflow channel.
The fifth channel is connected to the waste bottle via peristaltic pump which is running
backwards. Please read chapter “Mechanical Functionality of the HiClamp”.
The operation of the perfusion system is fully software controlled.
Do not start the perfusion without double checking the connected bottles and the waste bottle.
Warning: Spilled liquid can damage or even completely destroy the electronics of the
HiClamp. Please be careful when setting up the perfusion system and when starting the
perfusion of the washing station. Take care that inlet and outlet tubings are properly
connected so that flooding of the table and the mechanical components of the HiClamp
is efficiently prevented.
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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.
The basket with the oocyte inside moves in Y direction only, but is not moving during recording
minimizing recording artifacts and oocyte damage during transport.
The HiClamp is based on a innovative system in which the cell (or preparation) is exposed to
a test solution by moving it physically into the solution of interest, whereas in a standard system
the solution is applied on the cell. The provided solutions are disposed in a 96 well plate which
is fixed on the left part of the table. The cell (or preparation) is carried in the basket in the center
of the HiClamp. After adjustment of table complex and measuring head, the basket keeps
stationary while the table moves to immerge the cell in the desired solution.
12
For washing the cell between two measuring cycles there is a washing station placed
between the well plates.
HiClamp
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.
Immersion of the oocyte in the compound liquid is not sufficient to ensure a quick access of the
drug to the cell membrane. Passive diffusion of the molecule of interest alone to the membrane
would be too slow and mechanical stirring is therefore indispensable to ensure a fast access of the
compound to the membrane protein. Miniature stirrers were specially designed and conceived
with a proprietary structure to allow mixing of the solution in the 96 well format. Placing the
stirrers in the microtiter wells offers the additional advantage to reduce the volume of the well
and therefore to minimize the amount of solution required for the experiment.
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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.
14
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). The electrodes are lowered and the oocyte is impaled for recording (9).
Finally, the recording sequence starts when the oocyte is successfully impaled and an appropriate
resting potential is measured by both intracellular glass electrodes.
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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.
For example, the icon above symbolizes the loading plate that contains the oocytes to be
measured. For execution the information of this object, the HiClamp automatically loads the
next available oocyte, measures the resting potential and puts the oocyte back in its original
position if appropriate conditions, that means the holding potential is not met.
This icon based system offers several advantages that become evident when considering the
analysis. Data analysis is effectuated by a few clicks on the appropriate icons, which avoids
the learning of a specific language while offering the maximal flexibility. Results obtained
from different days of experiments are powerfully managed and merged by a module of the
software and results can be presented either as graphics or excel compatible data report sheets.
4.2 Installing the HiClamp Software
System requirements
Software: One of the following Microsoft Windows ® operating systems is required: Windows 7,
or XP (English versions supported with the NT file system). Other language versions may lead to
software errors.
Hardware: An USB 2.0 High Speed serial bus must be provided for the communication between
the HiClamp and the computer. 1 GB RAM working memory is strongly recommended.
Recommended operating system settings
The following automatic services of the Windows operating system interfere with the data
storage on the hard disk and can lead to severe performance limits. These routines were designed
for use on office computers, but are not very useful for a data acquisition computer.
Turn off Windows System Restore.
Turn off automatic Windows Update.
Turn off Optimize hard disk when idle (automatic disk fragmentation).
Deselect Windows Indexing Service for all local disks.
It is also not recommended to run any applications in the background when using the HiClamp.
Remove all applications from the Autostart folder.
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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. Virus scanners might cause a delay in the data transfer
when they scan each data package and file, which can then result in an instability of the HiClamp
program. If you need to install a virus scanner, please deactivate it before starting the recording
protocol. MCS cannot guarantee that a system that was modified by the user is fully operational.
Even data loss may occur.
Please check the system requirements before you install the software. MCS cannot guarantee
that the software works properly if these requirements are not fulfilled.
Important: Please make sure that you have full control over your computer as an administrator.
Otherwise, it is possible that the installed software does not work properly.
Installing the HiClamp Software with computer connected
The operating system and the HiClamp software are already installed on the provided computer.
However, you may need to reinstall or update the HiClamp software on the same or another
computer. Please check the system requirements if you are going to install the software on
another than the provided computer. MCS cannot guarantee that the software works properly
if these requirements are not fulfilled.
Power up the connected computer and wait until it is ready.
1. Double HiClamp Setup.exe on the installation volume. The installation assistant will open
and guide you through the installation procedure.
2. Follow the instructions of the installation assistant. The HiClamp software and all necessary
drivers will be installed on your computer.
4.3 Starting the HiClamp
To start the HiClamp, just switch on the data acquisition computer first. Press the I / O Power
switch on the rear panel of the HiClamp. The HiClamp is starting now.
Starting the HiClamp software
You may use the computer and the software offline, that is, the HiClamp is switched off,
to review and analyze your recorded data. However, if you start the software to operate
the HiClamp, the easiest way is to start the HiClamp first and then start the software.
Important: Please make sure that you have full control over your computer as an administrator.
Otherwise, it is possible that the HiClamp program does not work properly.
Double-click the HiClamp icon on the desktop.
- OR -
Select HiClamp software from the Start menu. The program starts.
Re-zero of the Axis
At that time the HiClamp table should move to the rightmost position. The re-zero sequence
of all the axis of the HiClamp will be done sequentially. The HiClamp is ready to start only when
this initialization phase ends and the program window appears with the play button visible.
This sequence takes approximately one minute.
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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. Please verify that the liquids flow regularly without the appearance air bubbles
in the perfusion chamber.
The Manual Window
The manual window is designed for the manual control of the solution, for placing the basket
into the washing station, for unloading the oocyte and for the cleaning of the basket. A single
click on the valve buttons switches on the desired pump. When all pumps are turned off, the
radio button for the value 0 is highlighted in blue.
The sliders and values on the right indicate the rotation speed of the inlet peristaltic pumps.
Note that the rotation speed of the outlet suction pump cannot be adjusted and keeps on
running at optimum as long as the HiClamp is switched on.
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