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Roboocyte2 Manual - Multi Channel Systems

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1. 77 Roboocyte2 Manual Start the recording in current clamp mode with Clamp Current 0 nA This will display the voltage of Current and Voltage electrode in green and blue respectively Uncompensated voltage offsets should be around 70 mV with electrodes filled with 3 M KCI Wait until the offsets are constant and Recording Clamp C Woltage Current Amplifier Control P o0 fo ce s00 Setas Defaut Recording Freg 20 Hz p stat a Stop Record Press the DC Offset button to compensate the electrode offsets The electrode voltages should be close to 0 mV after offset compensation 100 i Zo C 5 50 100 Sy z 200 100 z 10 15 ZU 25 30 Time fal Electrode voltages should be close to 0 mV and constant over time after performing offset compensation Move mto Liguid Liquid DL Offset Clear Offset H Press the Oocyte button to move the measuring head further down into the oocyte The Oocyte position can be defined in the Options menu Default value is 800 um which means 800 um above well plate bottom Oocyte Impalement 2 Up t Ss 20460 purr 78 Recording with Roboocyte2 Control Software If the impalement was successful you will notice a change in voltage on both electrodes for example the membrane potential of the oocyte is recorded 100 i T m Z a D 20 ae 100 a z 200 40 F 10 15 20 25 30 Time zl If the membrane pot
2. After the successful fitting the fit parameters Lower Asymptote Upper Asymptote EC50 IC59 and Slope Steepness are displayed 102 Analysis with Roboocyte2 Click on the button L2now stat Parameters J to open a window displaying the start parameters used for the actual dr fit SS 8 CF Used fit parameters J Lower Asymptote 11755 n Upper Asymptote 256 1 n ECSOMCSO 0 0369 mM Slope 1 46 When traces from more than one dr recording are displayed you can toggle between the z D9 0 individual dose responses EJ and fit the various dr data separately W Cuwe Dose Aesponse Curve UF Logistic Fit 4F Lower Asymptote y mint 2000 147276 n Upper symptote ymax 4000 2595 n ECS0 1C50 E000 A O 0372 mbl Slope S teeprniess eo ee 1 58 0000 Calculate Fit i Vary Far Estimate Start Parameters 12000 44000 fos E S _ Show Stant Parameters 0 01 01 j Show Start Parameters ATF Concentration rok 103 Roboocyte2 Manual Result Display IV Curve Similarly IV recordings can be displayed individually or in a set of IVs which can be plotted simultaneously multiple recordings 23 IV Curve Dose Response Curve a 15000 0 10000 5000 5000 Ae 0 5 10000 mms Il 5000 EF 15000 z 10000 a if E 20000 g 15000 a i 20000 5 25000 25000 30000 30000 35000 35000 40000 SLL 45000 45000 120 100 80 60
3. Click Home to bring the z axis and well plate carrier to the home zero position Click Coarse Position to move the well plate carrier to a position of the last xy alignment position Likewise the z axis is moved to the z position 3 mm above the last z alignment position Set Alignment saves the actual position of the carrier and the z axis as the alignment position Warning DO NOT press the Set Alignment button unless a proper alignment has been performed Change Plate will bring the carrier to the front of the Roboocyte2 where it can be easily reached to install or change a 96 well plate 48 Manual Mode Functions in the Plate Carrier Section All functions in the Plate Carrier section refer to functions of the carrier Plate Carrier Plate Nunc Standard 1286 not loaded Reference Home Park Pos Resist Pos well B Click the Reference button to start a reference movement of the well plate carrier After performing a reference movement the well plate carrier stops at a defined zero position Click the Home button to move the well plate carrier to the home zero position Click the Park button to move the well plate carrier to the park position Click the Resist Pos to move the well plate carrier to the resistance position Click the Well button to move the well plate carrier to the selected well Click the Air Cushion button to start and stop the air supply for the carrier Whe
4. Plate Carrier Plate Greiner Standard 128 Reference Home Park Pos Reference Home Park Pos Tube Tube Oocyte Liquid well a Air Cushion 7 t yx Ata e Fast Fast s Opm First perform an alignment before you click any button on the Manual Mode tabbed page Movement 47 Roboocyte2 Manual General Advice Warning Do not use any movement command without a valid alignment Always make sure that a proper alignment has been made and that you have not moved the carrier by hand or changed the TEVC probe afterward immediately before clicking any button Wrong movements of the z axis and the carrier can cause the glass capillaries of the TEVC probe to shatter possibly projecting splinters which can be dangerous Eye protection should be used and the Roboocyte2 s operation should never be observed up close with an unprotected eye The commands in this tab are mainly used to execute all kind of possible movements of the z axis and the well plate carrier The Movement tab is divided into different sections Global Plate Carrier Air Pressure Resistance Display and Z Axis Functions in the Global Section Global Reference Horne Coarse Position Set Alignment Change Plate Click on the Reference button starts a reference movement of the z axis and the well plate carrier After performing a reference movement the z axis and the well plate carrier stops at a defined zero position
5. AutoFill Concentration l Select concentration seres 7 2 5 10 20 50 100 200 500 mika 1 3 10 30 100 300 nh X Ok _ Aa Choosing the sequence 1 3 10 100 300 and the unit nM leads to the entries as shown below Configuration of Compounds per alve fl g Available Configurations Gaba DR O Valve Buffer Compound 1 Cone Unit 1 1 ORI GABA 1 nk 2 ORI GABA 3 nk 3 ORI GABA 10 nk 4 ORI GABA a nk 5 ORI GABA 100 nk E ORI GABA 300 nik T ORI GABA 1 phd g ORI GABA 3 pt g ORI GABA 10 phd 10 ORI GABA a pt 11 ORI GABA 100 pe 12 ORI GABA 300 ph Clear Complete Configuration Clear Configuration OF Selecte Complete the definition of all solutions and save the configuration with Exit y You have made changes to the input template configuration EmptyTemplate Configuration Save it under another name before you leawe this dialog After saving you can load the configuration at any time for later use either with Roboflow or with Gilson liquid handler or for any modification 64 6 2 6 3 Compound Application Using the Roboflow System The Roboflow System is simple easy to maintain and ideally suited for most rapid standard and expression tests dose response analyses and small screens The system consists of twelve valves and two peristaltic pumps The perfusion can be continuous but you can also pause the perfusion during the recording The small vol
6. be Well B12 Recs 7 0 E Row D wels 8 Recs 222 0 90 Analysis with Roboocyte2 Data List The selection in the Tree View section will determine the displayed wells and recordings in the Data List section Selecting the whole plate in Tree View will display all wells in the Data List Roboocyte p2 x 270171170 File Settings Help E H p2x2 20 21120 fells 13 Recs dij Results Settings Into Liquids Injection A Ron B Wele Re 0 7 ir well IE Series ROL Minimum PosMin Maximum Pos M Average Well BS Recs 7 0 emis oo Well B10 Recs 7 0 gt G Be 7 oo Well B11 Recs 7 0 b B10 7 os Well B12 Rees 7 0 e B11 7 H Row D Melle 8 Recs 222 0 t 1612 7 pe Well D2 Recs 7 0 t D2 7 p Well D3 Recs 7 0 t 03 7 Well D4 Recs 7 0 Well D5 Recs 7 0 b ODA Well D9 Recs 7 0 gone Well D10 Recs 92 0 e D9 7 Well D11 Recs 88 0 b D10 Well D12 Recs 7 0 bO B10 10 imam ran Selecting a row in Tree View will display all wells from this row in Data List view Roboocyte p z 270121120 Fie Settings Help E p2x2 20121120 TE 13 Recs 257 0 a R Settings Info Liquids Iniection Sow B fells recs 35 pe B ere J T a O well ID Series AOI Minimum Pos Min Maximum Pos M Aver Well BS Recs 7 0 e LIBS Well B10 Recs 7 0 e BS 7 oo Well B11 Recs 7 0 b B1017 n well B12 Recs
7. Open the pressure line and apply a pressure of 3 to 3 5 bar Warning Make sure that the compressed air used fulfills all requirements for working with the Roboocyte2 Otherwise use of an improper compressed air could result in physical harm to the hardware Especially check that the compressed air is absolutely free of water oil and any particles Refer to the chapter Important Safety Advice for more information Connecting the Roboocyte2 to the Power Line Finally plug the power cord into the AC power line input The diameter of the connector for the power supply is not round Please turn the flat part of the connector to the left when looking directly on the back side of the device Otherwise the connector will be damaged 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 12 Setting Up Hardware and Software 3 2 Setting up the Roboflow System robofiow E e malihira ig roboocyte s G m 6 The Roboflow System has been especially designed to work with Roboocyte2 The system consists of two peristaltic pumps and twelve pinch valves The valve pump for the compounds on the movable slide of the Roboflow manages the fluidic inflow the waste pump installed on the right side of the front panel is for aspiration of the fluids from the wells
8. multichannel systems Roboocyte2 Manual robo ecyte2 multichannel syste 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 2013 Multi Channel Systems MCS GmbH All rights reserved Printed 17 06 2013 Multi Channel Systems MCS GmbH AspenhaustraBe 21 72770 Reutlingen Germany Fon 49 71 21 90 925 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 Introduction About this Manual Welcome to the Roboocyte2 Important Safety Advices
9. 40 20 0 20 40 60 i Voltane my multiple recordings 46 lv Curve Dose Response Curve a 15000 0 10000 5000 0 10000 IIl 5000 15000 z 10000 Z 20000 5 15000 5 20000 5 25000 25000 30000 30000 35000 35000 40000 40000 45000 45000 5 Uis Zii Pie ee 120 100 80 60 40 20 0 20 40 60 bes ms Voltage mv If P n leak subtraction has been performed during recording you can select Leak Subtraction in order to subtract the leak currents from your recordings In the following example an lV recording has been performed with the test model cell TMC The TMC is mimicking the passive electrical properties of an oocyte and has therefore a voltage independent resistance R 1 Mohm and a linear IV dependency multiple recordings 6 lV Curve Dose Response Curve 40000 i amp 800 30000 alle 600 20000 iu 400 200 10000 is it j T 2 5 0 n C 200 a 5 f 5 10000 ee ae 600 20000 oe 30000 1000 1200 40000 1400 i z a Uy 150 100 50 0 50 100 l l Mene g Voltage m 104 Analysis with Roboocyte2 After selecting M Leak Subtraction ayp M Baseline Subtraction the IV dependency becomes corrected for the linear leak and becomes flat as expected for the passive TMC Note To perform leak subtraction baseline subtraction has to be selected too and the baseline cursors have to be placed properly multiple recordings 6 IV Curve Dose Response Curve S g
10. 40000 800 TE 600 20000 i 400 200 10000 Te 0 5 5 200 o is 10000 400 20000 Da 800 30000 i 1000 40000 4200 1400 4 AS TT eS i Lie eS eae eat epee NIL mer Sas Ui i ma tases eel l z a i a 150 100 50 0 50 100 os m Voltage m At the same time the large capacitive transients almost disappear as well No P 4 leak subtraction P 4 leak subtraction active multiple recordings 6 multiple recordings 6 40000 40000 30000 30000 20000 10000 10000 0 10000 10000 20000 20000 30000 30000 20000 Current n o IIL Current n 40000 40000 105 Roboocyte2 Manual Display Tools Scaling the Axes an oa 5800 20 nA SF m Similar as with the displays in the Roboocyte2 program you can scale and zoom the Data Display window with the buttons located below the display Likewise you can use the mouse to move or scale axes Move the mouse pointer above the respective axis the pointer will now appear asa hand n Hold down the left mouse button to grab and move the axis When you hold at the same time the control key keyboard the mouse pointer will change to an arrow and J moving the mouse or mouse wheel will rescale the axis Automatic Scaling Clicking this icon rescales the x axis to the whole time range and clicking this button E automatically scales the y axis to display the whole current range In addition to the trace all curs
11. Assembling the Tubing The measuring head of the Roboocyte2 carries an inlet and an outlet port for attaching the perfusion tubing The liquid is aspirated by the computer controlled aspiration pump and then collected in the waste bottle 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 Roboocyte2 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 The pinch valve system is easy to maintain because the liquid is only in contact with the tubing material and not with the valves Simply exchange the tubing in case of contamination You need silicone tubing ID 1 2 mm OD 2 0 mm for the pinch valves One set of preinstalled tubings is already provided together with the manifold for a quick start Please note that materials other than silicone are not flexible enough to seal tightly If you need another material for your application though you can use the silicone tubing for the short way inside the valve only and connect it to your custom tubing Use appropriate fittings which fit tightly Also regard the tubing dimensions specified and do not use a thicker or thinner tubing otherwise the valves may not
12. Record button is active the recorded data will be saved to the actual plate file Note A recording can only be saved when a plate file is loaded If no plate is loaded the Record button is grayed out and cannot be activated 52 Manual Mode The IV Protocol editor The lower part of the Measurement tab is dedicated to IV Voltage Step Protocol recordings Voltage Step Protocol Load Edit Create Protocol TMC Test saggi hes D 50000 0 Apply Stop Time ma 9 y t 100 o Hag a The display will show the IV current traces after loading and applying an IV protocol IV protocols are started by clicking the Apply button and stopped before finishing with the Stop button Use the Load button to open the Load Voltage Step Protocol dialog in which you can select an IV protocol Choose TMC test for example a predefined IV protocol in order to get familiar with the IV editor Load Yoltage Step Protocol Protocal TME test OneReconding OneRecoding 2 test VoltageSteps TMC VoltageSteps TMLT Leaks Sf i Lima I a a The name of the desired protocol is displayed in the Protocol window 53 Roboocyte2 Manual IV Protocol Click Create button to create a new voltage step protocol in the Voltage Step Protocol dialog or to apply a preliminary saved protocol The last used voltage step protocol is displayed in the Protocol Name window You can lo
13. ocuments and E E E A EAE a a E Scripts C Documents and Settings rettingertE gene Dateienshuli Channel Systems Roboocwte25 cripts Ee Liquids CADocuments and Settingesrettinger Eigene Dateien Multi Channel SystemssAoboocpytes Liquids eS DE File CA Documents and Settings rettingersEigen DateiensMultt Channel Systems Aoboocutes 201 2 Nov 15 acedb Cancel 3 Load a well plate already existing in the Roboocyte database Whenever you load or create a well plate it will be included into the Roboocyte2 database Then you can use the option Load Plate from Database to reload one of these plates Script Roboocyte Settings Load Plate Ctrl o Load Plate from Database h Mew Empty Plate Exit After selecting Load Plate from Database the Plate Selection dialog will open Plate seletion Database lc Documents and Settings rettinger Eigene Dateien Multi Channel Systems Roboocyte2 201 2 Noyv 15 acedb List of Plates Plate Date Comment R2 Version itr2 b 10 06 2011 09 41 22 1 3 1 12311 tr2 7 09 08 2011 13 39 37 1 3 1 12311 nachr 01 15 11 2012 11 45 51 1 3 1 12311 nachr 02 15 11 2012 11 58 37 1 3 1 12311 nachr 03 15 11 2012 11 59 43 1 3 1 12311 M Color Code for Plates found in Directory Roboinject Plates Roboocyte2 Plates Not found in Default Directories _ Load Cancel 40 4 2 2 Roboocyte2 Software Entries in red are well plates stored in the Roboinject folder b
14. 1 Name of compound 1 Vo 50 50 50 50 50 50 50 50 50 50 50 50 Date 1 10 10 2011 03 46 21 10 10 2011 03 46 21 10 10 2011 03 46 21 10 10 2011 09 46 21 10 10 2011 09 46 21 10 10 2011 09 46 21 10 10 2011 09 46 21 10 10 2011 09 46 21 10 10 2011 09 46 21 10 10 2011 09 46 21 10 10 2011 09 46 21 10 10 2011 03 46 21 D2 a Yo SS m m m m mm SS m SAA m 3 Analysis with Roboocyte2 Dat IDS Wo Dae3 ID4 Ma Yo Date 4 See SS eS Se ee eS eS ea Sea Ses Se eS The Injection tab includes information about the injection of the plate from left to the right Well The well number for the recording A1 ID The recording ID from 1 to 7 ID 1 Compound ID 1 Name 1 Name of compound 1 Vol 1 Volume of compound 1 Date 1 Date of the first injection ID 2 Compound ID 2 Data Display and Result Display The lower part of the main window of the Roboocyte2 software is divided in one setting section and two data displays The left window displays the result data traces in the right window you can toggle between an IV Curve and the Dose Response Curve The Setting section includes check boxes for the Baseline Substraction and for Drift Correction and Leak Substraction Select a Time Limit from the drop down menu O Dareia Subtraction inte Corector F Cuser n iano 180 a i g Wae DomArmporer Cune A Bl ul TTT TETTETETT mjo u 10 75
15. 7 0 g B11 7 E Row D wells 8 Recs 222 0 B12 17 H Well D2 Recs 7 0 Well D3 Recs 7 0 Well D4 Recs 7 0 i Well D5 Recs 7 0 oo Well D9 Recs 7 0 Well D010 Recs 92 0 well D11 Recs 88 D i well D12 Recs F D And selecting a single well in Tree View will only display this well in the Data List Roboocyte p2x 2701271120 File Settings Help E El pexe 2 211720 Wells 13 Recs 257 0 dij Results Settings Into Liquids Injection G Row B Wells 5 Recs 35 0 Well B5 Recs 7 i ii mel ID Series AOI Minimum Poshin Masmum Fos M Awer es b _ BS 7 2 Wel B10 Rees 7 D iwel B11 Recs 7 0 ke Well B12 Recs 7 D El Row D Wells 8 Recs 222 0 oo Well D2 Recs 7 0 Well D3 Recs 7 0 Well D4 Recs 7 0 oo Well D5 Recs 7 0 Well D9 Recs 7 0 Well D10 Recs 92 0 oo Well D11 Recs 88 0 i Well D12 Recs 7 0 91 Roboocyte2 Manual In order to have a closer look at the recordings in the Data List click on the triangle icon a Expand Collapse All 4 left to the well entry In this example seven recordings have been performed to generate a dose response curve for the P2X2 receptor that is an ATP gated on selective cation channel To expand all well entries simultaneously click on the icon Select Deselect All to select and display all recordings in the plate Another click on the respectiv
16. Documents and Settings srettingers gene DateiensMulti Channel Systeme Aoboocytes database O1 acedb Roboocyte Ini File CAD ocuments and Settings rettinger4nwendungsdatensMult Channel spstems Foboocytes oboocytes in Ix Roboinject Plates C Documents and Settings srettingersE igene Dateien Multi Channel Systems A oboinject Roboocyte Plates C Documents and Settings rettingersE igene DatetensMulti Channel Systems Roboocytez Plates User Defined Plate C Documents and Settings rettingerse gene D ateien Multi Channel Systems Aoboocytes backup Path Default Director when opening Plates from File System T Roboinject Plates C Roboocyte Plates User Defined Cancel 89 8 3 Roboocyte2 Manual Analyzer Displays In Roboocyte2 program you can either open a plate via File menu or by clicking on the Open Plate icon The selected file appears in the tree view Click the Expand button El to see the recordings Roboocyte Roboacybe _Plate lol E Fie elie Hai roboecyte2 Frobovcyted_ Plaid fetel lees 192 22 de endi Seting ino Ligue Irain pee iad ii D wa o p Hyra We Cate 1 In hace io Da Ir CE Thste 3 iDa Marad will AD Fisse 25 4 a BIT e SS Se S well AD fis 20 4 Al O Catpourd Hams aisom DO 1000371 ede i wel Ad Plocs 2 d AD 3 Compound hee ocom S Arhan ee o i Pree E fatale 4 Baer SU 16 AL o pami Haea Of TLS ee li Wiel G1 Fl
17. Fie Script Roboocyte Settings Tools Help eS F Use the different settings available in the menus to operate all functions of the Roboocyte2 software Some of them are also accessible via tool bar File Menu Script Roboocykte Settings Tools Help Load Plate Ctrl g Load Flate fron Database New Empty Plate Close Plate Exit The File menu allows to load a well plate from the database to load an empty plate and to exit the program Script Menu Roboocyte Settings Tools Help Load Script Chrl ShiFE O Save Script 45 Edit Script Ctrl ShiFE E You can load save or edit a script via the Script Menu Roboocyte Menu Fie Script Roboocyte Settings Tools Help Stark FS Alignment wizard Mowe to Parking Position Move ko Home Position You can start a script controlled recording open the alignment wizard or move to Park and Home position via the Roboocyte Menu 35 Roboocyte2 Manual Settings Menu File Script Roboocyte Settings Tools Help Options Change the paths for scripts plates or liquids and open the options window via the Settings menu Click on Directories to open the Set Directories dialog and define the different paths x Flate Files ERANT aLe and Settings srettingersEigene DateiensMulti Channel systems Aoboinject Scripts Liquids DE File C Documents and SettingsrethingerSE igene D ateien
18. Gilson Peristaltic Pump Perstaltic Purp Direction Foward C Backward Test and operate the peristaltic pump connected to the Gilson GX 271 in the Peristaltic Pump section Use the On button to switch the pump on and off use Forward or Backward radio button to let the pump turn forward or backward and use the Speed control to change the speed of the pump Pressing the Fast button accelerates the pump to maximum speed 59 6 1 Compound Application Linking Compound Applications to Recorded Data For later evaluation of your recorded data it is essential that all information about the applied compounds during recordings will be stored together with the recorded data The Roboocyte2 software offers a safe and convenient way to realize that with the Liquid Configuration editor The first step in liquid management will be to define buffers and compounds and assemble them into separate Liquid Configurations which you can use afterwards for your automated recordings To open the Liquid Configuration editor use the menu Tools Define Liquid Configuration File Script Roboocyte Settings Manual Mode Select Liguid Configuration Database Tool Define Liquid Configurations The Settings for liquid handling dialog will appear and you can choose between defining liquids for either the Roboflow Valve Liquids or the Gilson GX 271 Gilson Liquids Settings for liquid handling
19. The latter has the advantage of a less pronounced tendency to form salt crystals Wait a short moment until the tip has been completely filled Check this by using a microscope if necessary Backfilling glass electrodes F F 1 Insert the Microloader 2 Caretully Till the micro 3 Wait until the tine tip tip into the capillary electrode has been filled by capil as deep as possible lary forces There should be no air bubbles in the glass capillaries Otherwise the electrode resistance will be too high Gently tap on the glass to remove any air bubbles if necessary Take two of the chlorided silver wires out of the box stored in the small plastic bag and insert them into the glass capillaries Be careful and avoid that the coating of the wires is scraped away Residues of the coating material could possibly clog the capillaries 69 fe 8 10 Roboocyte2 Manual Click the alignment icon in the main window The Preparation window of the Alignment Wizard dialog appears Alignment Wizard i Preparation Plate Type e Te Tgteaeel Glare t cei pert Air Pressure Check a000 hPa Electrode Resistance ResstancePos Measure u Z AMIS Change Probe Pos Carrier Change Plate Pos Home Pos Next Cancel Click the button Change Probe Pos The z axis will move down a bit now allowing better access to the place where the measuring head electrode wires will be
20. This could lead to wrong movements of the Roboocyte2 which are potentially hazardous If you want to cancel the alignment leave this dialog box by clicking Cancel or Back The Resistance Test dialog box opens You can now test the electrodes resistance Please read the next chapter Testing the Electrode Impedance Testing the Electrode Resistance Before starting to do experiments with a new measuring head the electrode resistance of both glass microelectrodes should be checked After finishing the alignment the Resistance Test window of the Alignment Wizard appears Resistance Test Electrode Resistance ResistancePas 475 kOhm u 123 kOhm Back Finish Cancel 74 7 4 Recording with Roboocyte2 Control Software Click on the ResistancePos button to move the carrier to the Resistance Check Position and the measuring down In order to be able to perform a resistance check you have to place one of the small plastic dishes on the resistance position metal ring and fill it with oocyte Ringer solution After moving the measuring head to into the resistance position dish you can measure the electrode resistance by a click on the Measure button A fresh measuring head should have resistances of about 400 700 kOhms Click on the Finish button to bring z axis and well plate carrier to their home positions and you are ready to start the recording either in Manual Mode or automated by running a
21. backward direction Wait until the tubing is completely filled and no air bubbles are left Priming is completed 76 7 5 7 5 1 Recording with Roboocyte2 Control Software Recording Data Manual Recording Mode What You Can Do moving the measuring head into wells measuring the resistance of the electrodes determining electrode voltage offsets and compensate them impaling oocytes measure membrane potential clamp either current or voltage changing and testing various gain settings P gain gain crosstalk capacitance compensation CX switching on and off perfusion switch between different solutions recording and saving the data in continuous recording mode at various sampling frequencies recording IV dependencies What You Can Not Do Roboocyte2 Version 1 3 1 executing scripts saving the recorded IV dependencies linking solution exchanges to saved data from continuous recordings Manual Recording Step by Step Move the carrier to the respective well for example to well A1 This will only work if you have loaded or created a well plate before Move Carrier Home Move the measuring head down into the liquid wells should be filled with at least 200 ul of buffer Move mto Liguid DE Otset Clear Offset Check the resistance of both glass microelectrodes Resistances of a new measuring head should be clearly below 1 MOhm Resistance 4E2 kOh LI 510 k h
22. buttons on the right side to change the position In the Export Options window please decide whether you want to save the Table and or the Result in ASCII format and or the current Trace and or the Voltage trace Choose the Column Separator from the radio buttons Tab Delimited and others in Desired Characters Example Select Trace deselect Table and Results and define a filename by click on Browse button If you chose name for the filename the generated file will get the name name_Traces dat Click on Ok button will close the window and the selected trace is exported as ASCII text The ASCII columns are by default tab delimited but you can define other delimiters if needed The generated ASCII file will have the following format Column 1 contains the time in milliseconds column 2 contains the current values in Nanoampere Time ms Time ms _RecordingID 31 J nA _RecordingID 31 If you select more than one data trace for export additional columns will be generated with a separate time column for each trace Time ms _RecordingID 31 J nA _RecordingID 31 0 00 48 6 1 00 48 1 2 00 47 8 3 00 47 6 Selecting Voltage in addition will generate an additional column with the recorded voltage at the voltage electrode Time ms _RecordingID 33 J nA _RecordingID 33 U mV _RecordingID 33 Time ms _ RecordingID 33 RecordingID 33 J nA _ RecordingID 33 U mV 0 00 3 3 40 0 1 00 3 1 40 0 2 00 0 1 40 0 3 00 2
23. connected to Fix the measuring head on the cone of the z axis Use the provided forceps to plug the four connectors into the appropriate sockets on the axis Connectors viewed from the front and from left to right Current electrode left glass electrode bath reference for the current electrode RI voltage electrode right glass electrode bath reference for the voltage electrode RU Attach the perfusion manifold directly to the inlet nozzle Attach the tubings coming from either Roboflow or from the Gilson liquid handler to the measuring head The suction tube outlet is the coming out of the measuring head below the inlet tube 70 Recording with Roboocyte2 Control Software Installing the Well Plate Click the alignment icon in the main window The Preparation window of the Alignment Wizard dialog appears Alignment Wizard i Preparation Plate Type e Te Tateaeel t late t ci pert Air Pressure Check a000 hPa Electrode Resistance ResistancePos Measure u Z HS Change Probe Pos Carrier Change Plate Pos Home Pos Next Cancel Select Change Plate Pos to move the carrier to the front of the table Remove the used well plate if necessary Place the well plate which is defined in Plate Type in correct orientation on the carrier r aa r a ai e Fw a A Fw Pe SF p _ _ on ee ec i r 2 a CONE OG N Pla
24. dose response curve Robo2 TransmitCompoundValve valve is used in Roboocyte2 to generate the concentration axis of the dose response plot The two commands have to be inside of a recording for example between Robo2 StartRecording and Robo2 StopRecording Example Robo2 StartRecording Robo2 TransmitRecording REC_TAG_REF_COMPOUND Robo2 TransmitCompoundValve reference Robo2 Wait pre_agonist_s Robo2 OpenValve reference Robo2 Wait agonist_s Robo2 OpenValve 1 Robo2 Wait 10 Robo2 StopRecording 99 Roboocyte2 Manual When these conditions are fulfilled data will be plotted in the Result Display When you select one recording one concentration response value will be plotted Recording 30 Il Curve Dose Response Curve 0 4 a 5360 500 Ki 5370 1000 Ak 1500 Ilil 5390 2000 5400 T 2500 i Z sno S 3000 z 3 0 5 a 5420 3500 5430 4000 5440 4500 5450 5000 5500 5460 0 10 20 30 7 a as ee a a ATP Concentration mM E OY lew Se When you select all recordings belonging to one full dose response curve the corresponding number of data measured within the ROI will be plotted multiple recordings 7 V Curve Dose Response Curve 1000 g 1000 2000 ji 2000 3000 Iil 3000 4000 4000 g n 5000 e000 Z Eo 00 o 5 7000 oan 8000 8000 9000 9000 J 10000 10000 E Bra 11000 12000 12000 0 10 20 30 40 on a l AT
25. 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 MCS recommends 96 well plates from NUNC The oocytes are plated into the wells in a couple of minutes and can be kept for several days They quickly settle within the cone shaped wells and adhere to the well bottom after a few hours The oocytes do not have to leave the plate anymore you can easily transfer the oocytes from the incubator to the Roboocyte2 and back again The well plate carrier powered by linear motors hovers smoothly and noise free on a cushion of compressed air above the magnetic x y table The carrier operates at a resolution of 20 um The complete system does not require maintenance other than occasional cleaning of the steel plate The vertically moving z arm holding the TEVC probe is designed specifically for the demand of high speed and precision The z arm moves at a resolution of 10 um position and speed are computer controlled The status display of the Roboocyte2 shows the current state of the Roboocyte2 and the settings A quick alignment process guarantees that the oocytes are impaled precisely The ClampAmpC is a specifically designed digital TEVC amplifier and is completely automated Ready to use TEVC probes allow a quick and easy handling The ClampAmpC is an advanced version for the recor
26. impale the oocyte the arrow keys can be used to move further down in 50 um steps Finally the twelve valves can be individually switched on and off and the peristaltic pumps can be stopped and started by clicking the respective buttons Light Continuous Recording Window Continuous recordings will be displayed in the recording window the current trace in red and the voltage trace in blue To set the recording parameters please use the control windows right beside of the recording window Recording Clamp i Voltage f Current Current n Amplifier Control P 1000 4 100 a a a 1 D E cx S000 Set as Default Recording Freg 2o Hz p Start J Stop Record Time a gt co 9 jis t ao go Ha 2 3420 stm In the Clamp section you can switch between Current and Voltage clamp mode In the Set Clamp window you define the Holding Current in nA or the Holding Voltage in mV via up down boxes Click the Set button to confirm the clamp settings In the Amplifier Control window you adjust the amplifier parameters Please refer to the chapter TEVC Recording Background to find out more about how to optimize gain P and default 1000 and 100 respectively and capacitive compensation CX default 9000 of the digital TEVC amplifier Choose the Recording Frequency in Hz this is the sampling rate Clicking the Start and Stop button to start or stop a recording if the
27. protocol into the program Press the Load button in the Voltage Step Protocol section and select TMC test Voltage Step Protocol Load Edit Create Protocol __ ____ Load Yoltage Step Protocol Press Apply and the voltage step protocol will be executed The data display will now show the following Current nA Time ma Jg tima Hia S a a 28 Setting Up Hardware and Software Press the two autoscale arrows at the bottom of the data plot window s jt This will bring the current traces completely on the display 3 D Time ms co a ft 61320 nd H 673804 H GE a You can also zoom into a certain region by using the zoom box tool Current nA Time ma E k t B0300 n E2230 n ip a Complete zoom out can be accomplished by pressing the zoom out button a Al The current traces displayed now have been recorded with a proportional gain of 1000 nA mV and an integral gain of 100 1 s at a sampling rate of 20 kHz With the test model cell this leads to voltage clamp within less than 1 ms Similar values can be expected with a tight non injected oocyte 29 Roboocyte2 Manual Testing the Noise Level The current noise level during voltage clamp can be evaluated by recording from the TMC at different sampling rate and gain settings In the following holding currents at 0 mV holding potential are displayed to give you an impression ab
28. should perform backups of the Roboocyte2 data files rcd files and of the Microsoft Access database accdb at regular intervals and to appropriate 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 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 instructio
29. shows the general setup and the tubing connections of the complete Roboocyte2 and Gilson liquid handler configuration The solvent flow from Gilson tube to Roboocyte2 recording well is indicated by the blue arrows 20 Setting Up Hardware and Software Electrical Connections Please note that the Roboocyte2 needs to be connected to the Gilson GX 271 via the Gilson 508 Interface Module Setting up the GX 271 liquid handler GX 271 rear panel Gsioc connector Connect the z arm Roboocyte i here Connect the two solenoid valves of the transfer port station here GSIOC Cable Connect the Roboocyte to the 508 Interface Module here o Connect the Minipuls MP3 pump and the 508 Interface Module here Connect the Gilson z arm to the z arm port of the GX 271 Connect the two solenoid valves of the transfer port station to the solenoid valve ports Make sure that the cable labeled 1 is connected to the left and the cable 2 connected to the right port as shown in the preceding illustration Connect the RS232 port of the Gilson 508 Interface Module to the Roboocyte2 s serial port with the provided standard serial cable see Connecting the Roboocyte2 Connect the GSIOC port of the Gilson 508 Interface Module to the GSIOC socket of the Gilson liquid handler with the provided GSIOC cable Connect the Minipuls peristaltic pump MP3 to the same GSIOC socket of the Gilson liquid handler with the provided GSIO
30. the Start button in the Clean Valves section this will open all valves selected between First Valve and Last Valve for the specified Rinse Time At the same time both peristaltic pumps will be started at the speed selected After finishing the cycle all valves will be closed the valve pump will stop and the waste pump will continue to operate If you want to stop it you just have to press the Waste Pump button again 25 3 5 3 Roboocyte2 Manual Testing the Amplifier with the Test Model Cell You can use the provided test model cell TMC to test the integrated digital TEVC amplifier For initial testing you can measure the resistance and run a voltage step protocol This is also a good way to make your first experience with the recording features 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 Connecting the Test Model Cell Plug the current electrode connector red and the corresponding reference black of the test model cell into the sockets on the left side of the z axis red to front black to back Plug the voltage electrode connector blue and the corresponding reference black of the test model cell into the sockets on the right side of the z axis blue to front black to back Measuring the Electrode Impedance of the Test Model Cell On the Movement tab of the Man
31. the following standards The air must be absolutely free of water oil and any particles When air is compressed the humidity in the air is concentrated into a smaller area frequently condensing inside the air hoses Use a water separator to remove water from the air The provided water separator is not suitable to establish the necessary conditions it serves only as a control device If you regularly observe an accumulation of water in the provided water separator the compressed air does not fulfill the required standards This is extremely important Water in the system leads to corrosion and can destroy the device Use a filter with 5 pm pores or smaller to remove any particles from the air Set the external air pressure at least to 3 5 bars the maximum pressure of 5 bars must not be exceeded The actual operating pressure should be set to 3 bars by the included pressure regulator The Roboocyte2 needs a minimum flow rate of 20 I min when the carrier is moving Do not use other compressed air hoses than those provided that is ID 4 mm OD 6 mm from compressor to water separator and ID 2 5 OD 4 mm from water separator to the Roboocyte2 The use of other hoses could lead to a decrease of pressure Handling of the Carrier Do not try to lift the carrier off the x y table You may move the carrier carefully sideways by hand if necessary for cleaning purposes for example but not during operation If it happens that any liquid spill
32. voltage offsets Stop the recording in current clamp and switch to Voltage Clamp Chose 60 mV press the Set button and start the recording again Recording Clamp f Voltage C Current Amplifier Control F 1000 i 100 Cx 9000 Set as Default Recording Freq 20 Hz gt Start g Stop Record 27 Roboocyte2 Manual Now the voltage trace should exactly run at 60 mV and the holding current should be close to 600 nA because the oocyte membrane resistance of the TMC is equal to 100 kOhm If you switch to different holding potentials during the recording the voltage trace should be following immediately together with a change in holding current Recording s00 j Clamp Set Holding Voltage AO f Voltage tat 5 2 Wo Set gt 0 F f Curent a E _set D T E a Amplifier Control Lj ae T A00 E p 1000 4 I 1 Cs p 0 10 20 30 40 AO B0 Time fz Recording Freq 20 Hz gt tp 9 feos f 80n ena f sr mv 4 remy l Be a Stop o Record Voltage Step Protocol In order to test the step response of the built in amplifier it is best to perform a voltage step protocol with the test model cell For this purpose there is a pre defined voltage step protocol called TMC test First load the TMC test voltage protocol into the program Press the Load button in the Voltage Step Protocol section and select TMC test First load the TMC test voltage
33. work properly or may even be damaged Attach the tubing carefully without bending the silicone Please guide the tubes on there way from the manifold to the valves through the plastic tube holder to secure the correct attachment Do not insert the tubes to close they need scope for moving with the slide forward and backward Please control the provided manifold specially the connections of the provided tubes to the manifold 13 Roboocyte2 Manual Attaching the Manifold Please control the provided manifold specially the connections of the provided tubes to the manifold If you are going to use only some of the twelve tubes please connect the useless tubes to Ringer solution for example but do not leave the manifold connections open The manifold is fixed via magnetic forces to the housing of the slide of the Roboflow Adjust the manifold in a deviation of about 15 degrees to minimize the distance to the tube connection Attaching the Tubing to the Valves olf Twelve valves regulate the compound flow Only one valve may be open at a time Open a valve by hand or by a software command Take one end of tube which is connected to the manifold and insert it gently through the slot on the right side of the valve Press the valve with your fingertip to open it manually Do not insert the tube to close it needs scope for moving with the slider forward and backward Use the plastic tube guides to secure the tubings inside of the valve
34. z axis down to the park position Click the Tube button to move the z axis to the tube in the back of the carrier Click the Oocyte button to move the z axis down to oocyte position Click the Liquid button to move the z axis down to the liquid position Use the arrow buttons to move the z axis up and down either in Slow or Fast mode 50 Manual Mode 5 3 Manual Mode Measurement The Measurement tab page of the Manual Mode Manual Mode Movement Measurement Liquid Handling Move Carrier Move into Liguid Light fo to sell lan Liquid On J Home Valve Pump all 1000 Waste Pump al 1000 Recording Clamp C Voltage i Current Current n Amplifier Control P f 100 f o cx a00 _ Set as Defaut Recording Freq jo Hz b Start Stop 3 Record Voltage Step Protocol Load E dit Create Frotacal ______ Alu abeyoA Time fz Poa e ws Ble eS Sw oS Sm eS Current nA 1000 e000 S000 Time mz 4 4 100 10 Ana F Q Apply Stop The Measurement tab page of the Manual Mode is used to perform TEVC experiments manually Manually means that although everything is still controlled by software all steps like movements of carrier and z axis impalement voltage clamp solution exchanges can be realized with single commands under full control by the experimenter For different purposes it is useful to make e
35. 0 Min_Logistic4P 0 003 46 4773043957504 0 00336960372515171 100 532524488292 0 00378474308818542 163 920109674776 0 00425102813623059 238 117982912041 When you display more than one dose response curve and export the results additional columns and lines will be generated Dose Type of Fit Lower Upper Midpoint Slope Response Asymptote Asymptote Curve ID B8 0 Min Logistic4P 13634 3 147 7 0 033 1 57 B10 0 Min Logistic4P 11664 7 240 1 0 0389 1 55 B12 0 Min Logistic4P 11754 9 255 4 0 0369 1 46 Exporting Current Voltage Relationship Data If you display an IV dependency and export the data in ASCII format a file containing voltage and current values be generated U mV _FirstRecordingID 143 min nA _FirstRecordingID 143 130 48344 8601 120 37808 6629 110 28709 4663 110 9 1 Appendix Contact Information Local retailer Please see the list of official Roboocyte2 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 Roboocyte2 Mailing List you will be automatically informed about new software releases upcoming events and other news on the product line You can subscribe to the list on the MCS web site www multichannelsystems com 111
36. 000 F f z 200 f E 25000 D 95 Roboocyte2 Manual Data Display Whenever you select one or more recordings up to all recordings in the plate in the Data List the recorded data traces will be displayed in the Data Display Please see the screenshot above For example when you select a single continuous recording Recording 30 one current trace will be displayed Recording 30 500 1000 1500 2000 2500 3000 Current h4 3500 4000 4500 5000 5500 0 5 10 15 20 ed a0 5 Solution Exchange Tags in the Recording Display The small red numbered tags in the upper part of the data window reflect the time at which valves were opened In the example shown above valve 4 was opened and valve 1 closed at t 5 s and valve 1 was opened and valve 4 was closed at t 20 s Please note that these tags appear only when a single recording is selected Baseline Subtraction Drift Correction Leak Subtraction The left side of the data window offers three options Baseline Subtraction Drift Correction and Leak Subtraction Drift Correction and Baseline Subtraction Whenever you select a recording in the Data List the current trace is displayed in the Data Display together with cursors at positions defined in the recording script There are three types of cursor pairs drift cursors baseline cursors and the cursors for the region of interest ROI Please note that a drift correct
37. 11 Roboocytes Plate 10 10 2011 09 37 58 Plate for use with Roboocytes without 1 1 6 11311 DummyPlate 28 09 2010 15 34 43 Debug Dummy plate 1 1 6 11311 Click Create new empty database to create a new and empty database Create Database Enter Name of New Database Robooryte OF Cancel Click Change current database to switch to an other database lox Select from Available Database Files Roboocytez accdb bi Raboocytez accdb Roboocytez Scripts accdb TestS cripts accdb Cancel 45 5 1 Manual Mode About the Manual Mode Although the main purpose of the Roboocyte2 is the automated mode almost all functions can be accessed manually To open the manual mode click on the toolbar symbol a or use the menu command Tools Manual Mode The Manual Mode dialog consists of three different tabs the Movement the Measurement and the Liquid Handling that means each tab is dedicated to a distinct group of application Manual Mode Movement Click main menu Tools and select Manual Mode Or click the tool bar icon ia The Movement tab page appears Note Please use the manual movements of the Roboocyte2 for tests only Manual Mode l Xj Movement Measurement Liquid Handling Global Air Pressure Reference Home Check 3000 hPa G Coarse Position Set Alignment Resistance Change Flate i 5km u 123 kOhm 2 AMIS
38. 2 Manual Main Window of Roboocyte2 Software After starting the Roboocyte2 software you will see the main window with its four different sections ale x roboecyte2 Well Plate View Curent ret we E l E 40 seer beg j O 5 of ed g wi P D lt Cumt ret Script Log Display t E i amp Bi i d i Si a a a a a Tira lil Po 4 jos a F 10 0 a a r gil Soptfens Series WS D Connected The Recording display in the upper left window shows current traces during recordings which are saved in the r2d file All data between Robo2 StartRecording and Robo2 StopRecording in the running script The lower left window is the Control display showing all recordings performed All data between Robo2 StartControlRecording and Robo2 StopControlRecording in the running script Because this display shows also recordings which are not supposed to be permanently saved on disk it will also display voltage and current before impalement or during leak test routines to evaluate the quality of an oocyte before recording The part in the upper right is reserved for displaying the virtual Well Plate in which you can select oocytes that should be recorded The lower right section is displaying the script when the Script tab is selected or it is displaying the log messages when the Log tab is selected 34 Roboocyte2 Software The Menu and Tool Bar
39. 6 40 0 4 00 4 6 40 0 108 Analysis with Roboocyte2 Exporting Parameters from List View Table To export any of the parameters listed in the List View table select Table and move these parameters from left to right for example Well conc 1 unit 1 Minimum Pos Min After click on ok the ASCII file name Datatable dat will be generated Well conc 1 unit 1 Minimum Pos Min B12 30 M 5684 11685 B12 3 M 164 21920 B12 10 M 1107 13537 B12 30 M 4956 16349 Exporting Results Dose Response Curve Similarly you can export data from the Results Display for example dose response data or IV dependencies Select Results and all data shown in the Result Display will be exported This includes the current amplitudes as shown in the display which will be saved under the file name name_DoseResponseData dat C mM _B12 0 Min Imin nA _B12 0 Min C mM _B12 0 Min min nA _B12 0 Min 0 003 164 6675 0 01 1107 723 0 03 4956 9573 0 1 9501 1613 0 3 10928 1345 The fit parameters if a dr fit has been performed which will be saved under the file name name_FitParameters dat Dose Response Type of Fit Lower Upper Midpoint Slope Curve ID Asymp Asymp B12 0 Min Logistic4P 11754 9 255 4 0 0369 1 46 109 Roboocyte2 Manual The dose response curve calculated from the fit parameters above which will be saved under the file name name_CalculatedFit dat C mM _B12 0 Min_Logistic4P min nA _B12
40. C cables Connect the Minipuls peristaltic pump to the power line with the appropriate power cable Connect the power socket of the Gilson 508 Interface Module to the power line via the provided external power supply Connect the power socket of the Gilson liquid handler to the power line with the appropriate power cable 21 Roboocyte2 Manual Enabling the Gilson Liquid Handler GX 271 in the Roboocyte2 Program After installing the Roboocyte2 program enable liquid handler in the main menu Settings Options dialog box After changing the settings in the check box please close the Roboocyte2 software and start the program again to activate the communication with the Gilson LA Sti x Liquid Handling If Gilson Connected Positions of TEV probe Mowe to Liquid 2000 um Move to Docyte s00 um OF Cancel 22 3 4 Setting Up Hardware and Software Installing the Software Roboocyte2 The operating system Microsoft Windows 7 32 and 64 bit or XP with Service Pack 3 and the Roboocyte2 software are already installed on the provided computer However you may need to reinstall or update the Roboocyte2 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 If you want to access the same data files on a netwo
41. Default Directories Load Cancel The Plate Selection dialog displays the database specific plates which are stored in the selected database Because plate files can be located in different folders there is a color code which reflects the directory where the files are located RED Plates typically generated by the Roboinject program which are located in the Roboinject folder BLUE Plates in the folder which has been defined as Roboocyte2 Plate Folder in the Roboocyte2 program This is by default the Roboocyte2 plates folder MAGENTA Plates found in the user defined folder This folder can be defined in the directories menu of the Roboocyte2 program as described above BLACK Files not found in any of the three folders Analysis with Roboocyte2 Loading a Plate File from the File System Click main menu File Open Plate from the File System to select a plate file via browsing through your folders ffnen 2 x Suchen ir Plates Fi _Plate1 Plate Reenter Lateiname Roboocyte2_Plate2 rpt bung Dateityp Fioboocytez plate files rpf Abbrechen 2 Because only plates present in the database can be loaded this approach needs to know the location and exact the name of the plate file The folder which opens when you click on Open Plate from the File System can be selected in the main menu Settings Directories dialog Files and Directories Database File C
42. Directory far liguicds Esmaea aa Einstellungen Eigene DateiensMulti Channel Systems ARoboocyte sLiquids Configure Liquids with Valve Liquids Gilson Liquids Cancel 61 Roboocyte2 Manual Roboflow System Click the button Valve Liquids to open the Composition of Compounds per Valve editor for the Roboflow showing the default empty EmptyTemplateConfiguration Configuration of Compounds per alve i Available Configurations JEmptyTemplateConfiguation save Configuration save Configuration As Valve Buffer Compound 1 Conc 1 Unit 1 Compound 2 Cone 2 Unit 2 Compound 3 Cone 3 Unit 3 1 empty empty 0 mb empty 0 mbd empty 0 mbi f 2 empty empty 0 mb empty 0 mbd empty 0 rib 3 empty empty 0 mb empty 0 mb empty 0 mb 4 empty empty 0 mb empty 0 mbl empty 0 mbl Fi empty empty 0 mb empty 0 mt empty 0 mb B empty empty 0 mb empty 0 mt empty 0 mb Fi empty empty 0 mb empty mb empty 0 mb a empty empty 0 mb empty 0 mbd empty 0 rib g empty empty 0 mb empty 0 mbd empty 0 rib 10 empty empty 0 mb empty 0 mb empty 0 mb 11 empty empty 0 mb empty 0 mbl empty 0 mbl ies empty empty 0 mb empty 0 mbl empty 0 mb Clear Complete Configuration Clear Configuration OF Selected Valve Exit Cancel The editor assigns the 12 available valves to the respective compounds and concentrations used In addition you can select the buffer in which the compound is dissolved Before assembling the different solutions define y
43. IMPALEMENT STEPS I 8 Set IMPALEMENT STEP 50 Set IMPALENENT WAIT 1 al Set MIN INITIAL LEAKCURRENT 10000 Test Script r rirrmr rra rre m mair emm Break Save Cancel aes Create I Protocol Test Script Output For details please refer to the chapter Writing a script with the built in script editor Once you have loaded a well plate selected wells for recording and loaded an appropriate script the script will be executed as soon as you click on the Start icon below the menu bar The Database Tool As already mentioned all relevant information about the experiment is saved into a database file After installing the Roboocyte program the active database has the name Roboocyte2 accdb x Current Database Roboocyte2 acedb Show Plates Warming Changing the database also affects analyzing data because only the recordings which are present in the database can be analyzed Create new empty database Change curent database Close The database tool allows you to show the plates available to create a new empty database or to change a current database 44 Roboocyte2 Software Click Show Plates button to list the plates already measured Database EAD okumente und Einstellungen Eigene D ateiensM ulti Channel SystemssAoboocytes Aoboocytes accdb List of Plates Comment R2 Version fRoboocyte Flate 10 10 2011 05 37 58 Plate for use with Roboocytes without 1 1 6 113
44. Important Safety Advice High Voltage Requirements for the Installation Compressed Air Supply Handling of the Carrier Handling of the Z Axes Handling of the Ready to Use TEVC Probes Regular Backups Guarantee and Liability Operator s Obligations Setting Up Hardware and Software Setting up Roboocyte2 Hardware and Software Setting up the Roboflow System Setting Up the Gilson GX 271 Liquid Handler Installing the Software Roboocyte2 Testing the Roboocyte2 Testing the Movement of Carrier and Z Axis Testing the Roboflow Valves and Pumps Testing the Amplifier with the Test Model Cell Roboocyte2 Software Introduction Main Window of Roboocyte2 Software Well Plate View Loading a Well Plate Display Control Functions The Script or Log Window Manual Mode About the Manual Mode Manual Mode Movement Manual Mode Measurement Manual Mode Liquid Handling Compound Application Linking Compound Applications to Recorded Data Using the Roboflow System Using the Gilson Liquid Handler N OOO WOWOAWAWON NN NI 11 13 18 23 24 24 25 26 33 33 34 39 41 43 47 47 47 51 56 61 61 65 65 Roboocyte2 Manual Recording with Roboocyte2 Control Software Preparations for Recording Alignment Testing the Electrode Resistance Liquid Handling Recording Data Manual Recording Step by Step Automated Recording Step by Step Writing a Script with the built in Script Editor Analysis with Roboocyte2 The Roboocyte2 Analysis
45. P mM C 19 12010 160 H na Gg a The type of value calculated and plotted from the current trace within the ROI boundaries i depends on your choice You can select and plot using the icon S Minimum Value for Current 1 The minimum current value within the ROI boundaries for example for plotting peak responses in negative direction as shown JS Maximum Value For Current L The maximum current value within the ROI boundaries for example for peak responses in positive direction Area under the Current I Curve The area under the curve the area is calculated in consideration of the sign Therefore a careful selection of ROI boundaries and use of baseline subtraction is indispensable for significant results oo Average Value for Current I The average current within the ROI boundaries for example for extracting steady state currents o Extremal alue for Current 1 The extremum current within the ROI boundaries for example for extracting peak currents in positive and negative direction 100 Analysis with Roboocyte2 Capacitive Peak Currents multiple recordings 23 25000 20000 15000 10000 A Zr C SOU io z 5 E 5 m 2 a 5000 A R 0000 15000 20000 Sona ee 100 mg 0 E ala Minimum Value For Current tI ie io Mica x T xo l JN Maximum Value For Current I iii o eTEN z m 0 FaN i I 7 o gt Extremal Value for Current 1 dooa Below the Res
46. Software Analysis with Roboocyte2 Selecting a Database and Loading a Plate File Analyzer Displays Data Export to ASCII Format Appendix Contact Information 69 69 72 74 75 77 77 81 82 85 85 85 88 90 107 111 111 1 1 Introduction About this Manual This manual comprises all important information about the first installation of the Roboocyte2 hardware and software and about the daily work with the Roboocytez2 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 need information on the Roboocyte2 Scripting Language please refer to the Roboocyte2 Scripting Language manual If you are using the Roboocyte2 for the first time please read the important safety advice before installing the hardware and software Please see chapter First use of the Roboocyte2 where you will find important information about the installation and first steps Then start practicing using the chapter Recording in Manual or Automated Mode We offer you the opportunity of Learning by Doing which means that you start directly with practicing without much reading beforehand We suggest that you start the Roboocyte2 and then follow the tutorial step by step either using the integrated help or the printed manual Just decide what you like to do read all necessary information in short and put this information directly into practice Th
47. a base when the liquid configuration file is saved They are available either for Roboflow and for Gilson liquid handling Using Predefined Buffers and Compounds Once you have defined a set of compounds and buffers you can use them for assembling your liquid configuration either for the Gilson or for the Roboflow In the main dialog the Composition of Compounds per Valve editor you can choose buffers from the list of available already defined buffers by a click into the respective Buffer or Compound field Valve Buller Compound 1 1 oF _ emph 2 empty empty 2 empty empty 2 ORI empty a i empty J empty empty a ORI empty 400 DR a empty 4 emph empty 4 OR empty z empty empty z empty empty 5 ORI empty After selecting the buffer for example ORI for the first valve you can use the AutoFill option Please click with the right mouse button on the column header Buffer The Autofill function will be available Click on Autofill to enter the same buffer for the remaining eleven valves used In the same way you can enter and AutoFill compounds for the various valves Finally you assign concentrations of compounds to the valves If you use a compound with increasing concentrations you can use an AutoFill function for the concentrations as well 63 Roboocyte2 Manual A right click on the column header Compound opens the Autofill Concentration dialog for choosing the desired sequence of concentrations
48. ad different protocols from the Available Protocol drop down menu and confirm one of them with Load oltage Step Protocol E Protocol Name TME test Available Protocols Examplet Load Define Voltage Steps Leak Subtraction Tall alep l Enable VW m a eee E Incr j0 20 oO Rel Vo Leak Holdin OZ mw Type Standard wi Repeat 11 Add Column Remove Column Lett Right Wait Time between Sweeps TE 5 Baseline 5 m TE m P Gain Gain Sampling Fate Potential for Wait Time TE m pol EEZ r Tis a 3000 100 20000 Hz 100 AO 0 H0 100 z 10 15 20 25 30 Time rms Save and Accept Cancel In the Define Voltage Steps window the voltage step protocol is defined Define Voltage Steps Type Step Step 2 Step 3 mmo a pooo Repeat 11 Add Column Remove Column Lett Right wait Time between Sweeps 1 Baseline 5 ms T Potential for wait Time TE mO py ip se 1 54 Manual Mode The example TMC test is composed of three columns starting with a step from 0 mV to 100 mV and back to 0 mV to vary the number of columns please use the buttons Add Column and Remove Column The duration for each step is 10 ms There are 11 repeats with an increment of 20 mV which ends up in a final step to 100 mV The relevant segment for the voltage versus which the current will be plotted can be chosen with the check box Rel Volt If the
49. ase Info DB Version 87 8 2 Roboocyte2 Manual Selecting a Database and Loading a Plate File In Roboocyte2 data acquisition software a raw data file is generated r2 during recording from a well plate file and all information gathered during the recording is stored in the active database In order to analyze data you have to select the respective database and load the plate file that has been recorded into this database When you start the Roboocyte2 program for the first time the database presently active in the Roboocyte2 program will be selected automatically As soon as you select another database in Roboocyte2 this database will be remembered and loaded automatically when you start Roboocyte2 next time Open a Plate from the Database Click main menu File Open Plate to select a plate file directly The dialog Plate Selection appears Database C Documents and Settingesrettinger Eigene DateiensMuli Channel Systenms Aoboocytes database 01 accdb List of Plates Plate ID Plate Date Comment Re Version a a eee oe Seca coe Sap ae n Ch O2 11 12 2012 14 01 52 1 3 1 12338 Pae2 compound screen U5 11 12 2012 14 02 24 1 3 1 12338 Nak ATPasze 013 11 12 2012 14 03 19 1 3 1 12338 GAT1 OF 11 12 2012 14 03 04 1 3 1 12338 TRFY O1 11 12 2012 14 02 50 1 3 1 12338 PH test 11 12 2012 14 07 23 1 3 1 12338 Color Code for Plates found in Directory Aoboinject Plates Roboocytes Plate User Defined Not found in
50. c start the valve pump a so Usually the holding current gets smaller when the perfusion is on see figure above where the perfusion was started at about t 36 s Whenever you want to save the actually recorded data press the Record button The appearance of the red light shows that recorded data are saved ZO E 10000 Ta 20000 2 30000 0 50 100 150 Time 3 Switch between different solutions to activate inhibit modulate or deactivate endogenous or expressed ion channels receptors or transporters The figure shows activation and deactivation of the P2X2 receptor an ATP gated channel with 3 10 30 100 300 and 1000 uM ATP Voltage Step Protocol Load Create Protacal TMC test Apply Stop 80 Recording with Roboocyte2 Control Software In addition you can execute IV protocols to work with voltage gated ion channels or test the voltage dependence of ligand gated channels or transporters For details about how to define IV protocols please refer to the respective chapter of this manual The examples below show IV recordings from an oocyte expressing P2X2 receptors in presence and absence of 30 uM ATP Please note the different scaling of the current axis Peer reer rere rere rrr rrr rrr rrr 100 200 S00 Time me Time me 7 5 2 Automated Recording Step by Step In the previous chapter the manual recording abilities of the Roboocyte2 have been presented All thes
51. ce the well plate onto the carrier and press its side against the springs in the lower middle and lower right corner of the carrier Then put the rest of the plate down onto the carrier The well plate position is correct when there is no play between the well plate bottom and the carrier surface at all edges and when the well plate has full contact to the left and upper side of the carrier holding frame Important The well plate should be immovably fixed on the carrier otherwise the impalement of the oocytes will not be successful 71 7 2 Roboocyte2 Manual Alignment You have to align the position of the carrier relative to the measuring head i e the electrode tips under microscopic control The Roboocyte2 always moves relative to a standard position The aim of the alignment process is to define this standard position If this is not done properly the Roboocyte2 moves to wrong positions The alignment process also compensates for minor changes in the dimensions of different well plates and variations in the length of the electrodes To achieve a stable recording it is essential to perform an alignment each time before you start a recording sequence Note The alignment is very important for obtaining best results An alignment is strongly recommended each time before you start recording It is very important that the glass capillaries are centered on the alignment device Otherwise the oocytes are not properly hit Warning Hitting th
52. cify a rectangular region in the data display to which you can zoom if Use the Reset button to get back to the original view Exporting and printing of the display ici When you click on the Plot icon additional symbols will be displayed which allow you to save or to print the traces on the display y Click the Copy to the Clipboard icon to send the actual display picture to the clipboard Click the Save button to save the display in different kind of formats for example jpeg tiff png etc ty Click the Print button to print the display Click the Preview button to have a preview before using one of these functions Using the tracking function j If the tracking function is selected the x axis moves during recording together with the recorded current or voltage traces 42 Roboocyte2 Software 4 2 3 The Script or Log Window Like the well plate view window the script or log window is empty after the program start This changes after loading a script which can be done by using the menu Script Script Roboocyte Settings Tools Help Load Script Ctrl ShiFk 0 Save Script 45 Edit Script Ctrl ShiFk E Script files are stored in the folder specified during the first run of the software by default in the Script folder After loading the script it appears in the Script tab of the Script or Log window During execution of the script the actual position in the script w
53. cripting Language manual or to the help 2 1 2 1 1 2 1 2 Important Safety Advices Important Safety Advice Warning Make sure to read the following advice prior to installations of the Roboocyte2 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 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 cha
54. de icon to open the Manual Mode window For details fat Click on the Home icon to move the carrier to the home position Well Plate View Loading a Well Plate Directly after the program start the well plate view section is empty This changes after loading a well plate into the program Loading a well plate is obligatory to perform measurements on oocytes Without loading you will not be able to move the measuring head into a well and or an oocyte to perform recordings There are three different ways to load a well plate 1 Creating a new empty well plate Chose New Empty Plate from the file menu to create and load a new well plate Script Roboocyte Settings Load Plate Chri ca Load Plate From Database New Empty Plate h Exit The well plate will be stored in the Roboocyte2 Plates folder 39 Roboocyte2 Manual 2 Load a well plate generated by the Roboinject program Chose Load Plate from the file menu to load a an existing well plate Script Roboocyte Settings Load Flate Chrl 0 Load Plate From Database Mew Empty Plate Exit The default folder from which you can chose a well plate file is the Roboinject folder which has been created when you installed the Roboinject program If you are not using Roboinject you will be redirected to the My Documents folder You can change the default folder according to your preferences via the Settings Directories menu Set Directories X Seto eee
55. ding of fast sodium channels with a faster rise time and a higher sampling rate Roboocyte2 Manual The integrated pinch valve Roboflow System is an ideal perfusion system for drug receptor characterizations and quick expression tests The Roboflow is computer controlled and works virtually pulsation free thus minimizing noise during recording E gt Jar I Sj Lee For high throughput screens the Roboocyte2 uses the advantages of an industry standard liquid handler from Gilson optional The Gilson liquid handler version GX 271 is supported a The use of the GX 271 liquid handler is an optional feature of the Roboocyte2 Test compounds are loaded onto the trays and aspirated by the Gilson probe The liquid is transferred to the recording well via a peristaltic pump A transfer port gives access to one or two larger medium bottles for example for frog Ringer s or an often used reference compound Introduction The Roboocyte2 Software Operate the Roboocyte2 collect and evaluate the data by using the Roboocyte2 software The easy to use graphical user interface of the Roboocyte2 software makes daily work with the Roboocyte2 quick and easy Recording is started by a single mouse click The Roboocyte2 controls the run for all 96 oocytes automatically even including a wash cycle Thus the recording can go on overnight unsupervised You can define different experimental setups not only for different well plat
56. displayed in the Data Display On the right side you can toggle in the Result Display between the automatically generated IV Curve or a Dose Response Curve The main menu bar holds three options File Settings and Help Main menu File Roboocytez File Settings Help Open Plate 1 Cia Roboocytes Plates Roboocyte Plated pr 2 CH WRoboocyte Plates Roboocyte Plate rpr 3 Ch Multi Channel Systems iRoboinject Roboocyte Test rpt 4 CH WRoboocyte Plates DurnmnyPlate rp Open Plate From File System Close Plate Export Exit 86 Analysis with Roboocyte2 Menu to Open Plate to introduce a Roboocyte2 raw data file rpf into the analysis software The last used plate files are displayed in the second row of the menu Menu to Export data in ASCII format and to Exit the program Please see chapter Selecting a Database and Loading a Plate File for details Main Menu Setting S Roboocyte2 Settings Directories Select Database Menu for setting the database path and for selecting the database Please see chapter Selecting a Database and Loading a Plate File for details Main menu Help Roboocytez Plate File Help eS About Roboocyber Click About Roboocyte2 to get information about the Roboocyte2 software version and the database x roboecyte2 Roboocwe Version 1 4 1 13018 Copyright 2013 Multi Channel Systems MOS GmbH Datab
57. e Considering a volume of 200 ul in the well the solution should be exchanged after about 2 s Please refer also to the Gilson Minipuls Manual which describes the relationship between tubing ID and flow rate For information on automated operation of the liquid handler please refer to the Java scripting language manual Please see the Minipuls 3 Peristaltic Pump User s Guide and the GX 271 Liquid Handlers User s Guide from Gilson if you have questions concerning the general operation of the peristaltic pump or the liquid handler respectively Using the Transfer Ports of the Gilson GX 271 Gilson GX 271 transfer port station Please note that the information in this chapter Using the Transfer Ports applies to Gilson model GX 271 only Transfer ports can be used for aspirating solutions from large reservoirs or bottles for example for often used solvents such as Ringer or a reference compound The transfer port lines are closed by solenoid valves when they are not in use to avoid that either liquid drips out or air is sucked into the transfer port lines and to avoid salt crystallization inside the port Please keep in mind to include the appropriate valve control commands in your protocols See the Roboocyte2 Scripting Language manual for more information The GX 271 rinse and transfer port station combines two transfer ports with a drain and rinse station e g for emptying the tubing at the end of the working day or for rinsing the needle F
58. e alignment device can cause the glass capillaries to shatter possibly projecting splinters which can be dangerous Eye protection should be used and this alignment should never be observed up close with an unprotected eye Place the microscope in a position onto the carrier table which allows to focus on the alignment device plugged in well H12 Alignment Device Click Alignment Wizard in main menu Tools Or click the icon in the main window The Preparation window of the Alignment Wizard dialog appears Alignment Wizard Preparation x Plate Type EERE EEE Air Pressure Check S000 hPa Electrode Resistance ResistancePos Measure u Z Aas Change Probe Pos Carrier Change Flate Fos Home Pos Next Cancel Click Change Plate Pos to install the alignment device on well H12 72 Recording with Roboocyte2 Control Software Click next to open the Alignment window and the carrier will move to the x y coordinates of the last alignment position At the same time the z axis will move down to the z position 3 mm above the last alignment position Alignment ey Plate Carrier ey 123670 pm Y 59203 pm Place the microscope in a position onto the carrier table which allows you to focus on the crosshair engraved into the surface of the alignment device Move the z axis and the carrier until the electrode tips approach the cross hair by pressing the respecti
59. e icon works in the opposite direction Data List Results tabbed Page dij Results Settings Info Liquids Injection i well ID Senes ROIL kinima al A3 8 Ww A3 OTB T 0 OF iv AS 17 T 0 555 iw A3 18 T 0 557 w A3 15 T 0 F28 w A3 20 0 0 H28 AS 4 0 0 HAr Ww A3 22 0 0 H20 Ww AS 23 U U H20 al A3 4 VoltageSteps TMCS Ww AS 76 0 0 214745 wo AS 81 U 0 214r 4a Ww AS 86 U 0 214r 4a wo AS g 0 0 214r 4a The Results tab includes the following information from left to right Pos Min Masimum S080 S080 S200 geri 5045 S070 4390 5030 D O O A Well The well number for the recording B12 ID The recording ID from 30 to 36 Bon oe oe J27 17 oF 1547 aieia 214 46 214 46 214 46 214 46 Pos M Average Area 4565 5040 4600 5150 5075 SUSU 5075 s08 D O O A D68 Ba 556 Ha 44 316 54 24 Fal 529 Hed 425 SS Sa aa a a Se A Lett 140 140 140 140 4340 140 140 145 145 145 145 Right eal 230 200 eal 230 gri ea 2al eal eal eal ea Series The series number if no series has been assigned in the recording script series value is zero ROI The ROI Region of Interest number Minimum The minimum current within the ROI Pos Min The position time within the ROI where the minimum was detected in ms s or min Maximum The maximum current within the ROI Pos Max The position time withi
60. e printed manual and 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 Roboocyte2 help Check also the Multi Channel Systems MCS GmbH web site www multichannelsystems com for downloading up to date manuals Roboocyte2 Manual 1 2 Welcome to the Roboocyte2 roboocyte2 The Roboocytez2 is a fully automated all in one solution for high throughput screenings of ligand gated and voltage gated ion channels as well as electrogenic transporters based on the standard Xenopus oocytes expression system Main Features e High throughput of experiments e Recording of 96 oocytes over night e 24h operation without supervision e Automated TEVC recording e Voltage gated ligand activated channels and electrogenic transporters e Flexible design of automated recording sequences e Automated compound application e Automated cell wash Introduction Operation Summary The integrated TEVC amplifier operates in voltage and current clamp mode Inst
61. e single manual steps can be performed automatically for one cell after the other by using a recording script To start an automated recording you have to prepare the Roboocyte2 and the software as follows 1 Place the oocytes into the well plate or use a well plate with injected cells 2 Put the well plate onto the carrier 3 Prepare and install the measuring head 4 Prepare the solutions and prime the tubings in order to get rid of any air bubbles 5 Define or select a liquid configuration that meets exactly the liquids used 6 Make an alignment of the measuring head to the alignment tool 7 Load the right script 8 Select the oocytes to be measured 9 Start the script recording Please refer to the Scripting manual for details about how to write a script 81 7 6 Roboocyte2 Manual Writing a Script with the built in Script Editor Please load a script via main menu Script Load Script After loading a script you can review or edit the script with the built in script editor Script Log ff LTRecordingTestli 4s ff Copyright 2011 by Multi Channel Systems MCS GmbH Robos EnableRuntimechecks false Robo2 Log Sceript SCRIPT FILE for war i UO i lt Robhos SelectedWells Count i i Robod SetHoldingturrent 0 Robo2 SecCurrentClarmp i Robodg SetHoldingVoltage 20 El C Dokumente und Einstellungen Eigene Dateien OK Click on the icon E at the lower left cor
62. ead of having a vast amount of different pull down menus there is one clearly structured menu bar and also a toolbar presenting commonly used commands Therefore you will quickly become familiar with the Roboocyte2 The first step is to set up your virtual well plate according to your real well plate and the experiment The virtual well plate is visualized in a 12 x 8 grid as the real one and therefore it is easy to overview and to handle For managing high throughput compound screens enter compounds and concentrations in the Liquid Handling spreadsheet This information is linked to the corresponding data traces and filed into the database Select the oocytes that you want to record or load a customizable file Then simply start the recording sequence by mouse click The run will proceed automatically until finished or interrupted by the user Information about the plate and the current state of all oocytes is displayed and updated online on the screen You may save the well plate and load it later to proceed with your experiment or to review and analyze your data Basic analysis features and export filters are included for example P n leak subtraction graphs The Roboocyte2 Hardware The Roboocyte2 is compatible with standard lab equipment and can be easily integrated in your working environment Software controls for adjusting the amplifier replace any knobs on the device The Roboocyte2 is straightforward and easy to operate
63. ece Al JO Compound Haea O TANT AEF 0 n ee Al d Compound Namecfcom S0 TAINAN 0N 0 0 WAIBA Place Xil AlI A empardi Heada B 1002 een o i i AI G Corp Haea D WSR ae i F AI 7O Compari Haaa i O TA a T i b OA 20 VollegeStepe_ THE b Oar I A 20 ViekegeStepe TMS Om b Ad 2 VokegeStepr_ TMC p CAs p Ad aV TAC EON piigi THE b O Bi ay Vokagediepe_ TMCS k Aa 20 RI E a TMS D4 povp TACI Tree View When you load a well plate basic information about the plate recordings will appear in the Tree View window pee 2 21120 wels 13 Recs 257 0 The plate name for example p2x2 20121120 the number of wells of oocytes respectively from which successful recordings have been performed 13 the number of recordings 257 and the number of leak recordings 0 Click on the Plus icon to list the rows from which recordings have been performed El p x2 20 21120 Melle 13 Recs 257 0 H Row B wells 5 Recs 35 0 H Row D ells 8 Recs 222 0 Again basic information is displayed 35 recordings have been performed from 5 wells in row B and 222 recordings have been performed from 8 wells in row D Click on the Plus icon for each row to open the view on the single wells again showing the information about the number of recordings E psz 20121120 wels 13 Recs 257 0 Row B welle 5 Recs 35 0 os Well BS Recs 7 O Well B8 Recs 7 0 Well B10 Recs 7 0 Well B11 Recs 7 0
64. ential of the oocyte is not detected on one or both electrodes move the measuring head stepwise down using the arrow keys Stop moving the measuring head as soon as you record the membrane potential on both electrodes Oocyte Impalement 2 Up t 3 20460 purr Both glass microelectrodes record the oocyte membrane potential after proper impalement i 0 S nt 50 T 3 a 5 H0 100 z 10 15 20 25 30 Time fal Now after checking the electrode resistances performing offset correction and stability check as well as successfully impaling the cell you are ready to start the recording under voltage clamp Recording Clamp f Voltage C Current Amplifier Control F 1000 l 100 Cx 9000 Set as Default Recording Freq 20 Hz Stop Record 79 Roboocyte2 Manual Stop the current clamp recording and start the recording under voltage clamp by selecting Voltage instead of Current and press Start a S F al 40 El 3 A0 ED E0 0 20 40 E0 ol 100 120 Time The display will show the voltage of 60 mV if 60 mV is selected and the corresponding current the size of which is dependent on the membrane resistance of the oocyte for example only tens of nanoamps for a good oocyte Valves 10 29 30 40 50 60 a E al 4 al 10 a 11 a 12 al Pump 2 a Check membrane current with perfusion Start the perfusion in this sequence a start the waste pump b open the valve
65. es but also for specific selections of wells on the same plate A P n leak subtraction and several control features to save time and compounds are provided Start a wash cycle after a preset time period to maintain a high viability of the cells Automated analysis features are included in the Roboocyte2 software but you can export the data to your custom evaluation software as well You can graph the data and generate reports with the Roboocyte2 software The large amount of data generated by the Roboocyte2 can be managed with a Microsoft Access 2010 database not included The database can be accessed over a network by multiple users How is this high degree of automation and flexibility for such a demanding task achieved This is possible by using scripts small text files containing commands Users can write their own scripts with any basic text editor or with the editor embedded in the Roboocyte2 program All experimental settings are defined within the script You can write a script for any kind of experimental setup Once the appropriate script is loaded into the Roboocyte2 software simply click the Run button to start the robot The script is then performed without the need for further customization and supervision The Java Scripting Language has been specifically designed for the Roboocytez2 It allows the full automation of various applications For further information on the Java Scripting Language please refer to the separate Java S
66. hown in the last figure a leak current of about 70 nA remains after drift correction Activating the baseline subtraction subtracts the baseline current and the current trace runs close to 0 nA at the baseline cursor position Without Baseline Subtraction With Baseline Subtraction Recording 30 Recording 30 Current n Current n meea a a Ee LS a 5000 10000 15000 20000 25000 30000 5000 10000 15000 20000 25000 30000 ms ms For performing baseline subtraction the average current between the baseline cursors is calculated and subtracted from the current trace 97 Roboocyte2 Manual Changing Time Unit To change the time unit of the result display please use the Time Unit drop down menu Time Unik mz Cursor Tools On the right side of the Result Display you find a number of buttons which refer to the cursor tools Click the Set ROI limits icon A and the Edit ROI Cursors dialog appears Edit ROI Cursors x Left Right Baseline 2055 ms 3220 ms Araylsis b660 m 13000 m Drift Corr 15091 m 18093 ms OF Cancel Now you can set the left and right positions of the Baseline Analysis and the Drift Correction manually Click the Zoom to fit icon Ql to zoom all data into the data window Click the Retrieve ROI s and Drift Cursor icon alle and all cursors will return into the data window for example after zooming in Click the Show Hide Baseli
67. ill be highlighted and built in or user defined log messages will be written in the Log tab of the Script or Log window Script Log Roboe Log Seript SCRIPT FILE Roboz szetDialogvariable WashoOnstart 0 washonstart on 1 off 0 Robo2 Showbialogi vet AMPLIFIER GAIN F 1000 vet AMPLIFIER GAIN I 100 vet DOOPFSET RANGE 3 Het DOOFFSET DELAY 15 Het DOOFFSET WAIT S Set MIN RMP 8 Set IMPALEMENT STEPS Uit Set IMPALEMENT STEPS 1 8 Set IMPALEMENT STEP 50 Set IMPALEMENT WAIT 1 Ej KE usersiAobo 2 working customer scripts aglerche scriptss ECNOGS KCNO pnleakjs Script Result Once loaded you can edit the script either by clicking on the script edit symbol in the lower left corner of the Script or Log window or by using the menu command Script Edit script Roboocyte Settings Tools Help Load Script Ctrl ShiFk 0 Save Script 45 Edit Script Ctrl ShiFk E 43 Roboocyte2 Manual The script editor will open Edit K users jr Robo 7 working customer scripts aglerche scripts KCNQ KCNQ_pnleak js File Edit Search Script Robo2Z Log Script SCRIPT FILE Robo SetDialogVariable WashOnStart 0 nashonstart on 1 ofe o ys Robo2 ShowDialog Set AMPLIFIER GAIN P 1000 Set AMPLIFIER GAIN 1 100 Set _DCOFFSET_RANGE 3 Set _DCOFFSET_DELAY 15 Set_IDCOFFSET_WAIT 5 Set MIN RMP 8 Set IMPALEMENT STEPS U 4j Set
68. in the script In the example above ControlDisplay SetVAxis 0 30 was used instead of ControlDisplay SetXAxis 0 30 Please use the test function always after writing or editing a script Another great advantage of using the built in script editor is the Code Completion List Box which is a great help in writing scripts Whenever you start to type a command like Robo2 this function opens a list box with all possible commands starting with Robo2 72 i RecDisplay Clear i T3 en 74 Roboz Fo t PrintSkandard ariables al 76 iF OOC Questio TI ff a y R cea errrerere CON POUND es ee J aeda e aaa coc Lo REC_TAG_REF VOLTAGE oO REC_TAG UNKNOWN Sl for vy REC_TAG VOLTAGE Counts i De i t RecordIYProtocol Oo o va oS Referencesy i o4 Y References as aie uis bd 56 OT Controlbisplay Clearti ict RecDisplay Clear 1 Bo a0 fy Measuring head moves to selected well 83 Roboocyte2 Manual When you continue typing for example Robo2 Sto the command is selected in the list box and you can select it with a mouse click without typing the last letters 72 i RecDisplay Clear i 73 Robo g 5etsampleRate SANPLERATE 74 Rohoe Sto Fo oS SetvolkageClamp al T ff OOC Setwellnfo ti if l q Showpage eee eee FO Roboz ShowStandardDialog 9 l A StartControlRecording ou Y SkartRecarding i Sl for V StartTimer Count i B2 i taponi 53 Wa mp
69. ined the product from a different source other than Multi Channel Systems GmbH Installing the Transfer Port on the Locator Pane Please see also chapter Using the Transfer Port Important Please note that only the leftmost rinse drain and transfer ports can be used with the Gilson GX 271 liquid handler The other transfer ports have to be closed with plugs you can use convenient screws Do not remove the plugs otherwise the suction will not suffice to aspirate solvents from the transfer ports Connect the provided waste tubing to the waste outlet on the back of the rinse transfer port Station Remove the two Phillips screws from the back of the rinse transfer port station Raise the cover on the rinse station and align the holes in the rinse transfer port station with the holes on the top right side of the locator pan please see figure Gilson GX 271 Solvent Deck which shows how the Gilson should look like after the installation The cover does not need to be removed completely Locate the two Phillips screws included with the rinse station and place them through the holes on the base of the rinse station see figure Tighten the screws using a Phillips screwdriver Lower the cover on the rinse station and replace the two screws on the back 18 Setting Up Hardware and Software Gilson GX 271 Solvent Deck Rinse transfer port station Locator pane Empty rack slots General Setu
70. ion The cords should be plugged in without the use of excessive force The following photo shows the rear panel of the Roboocyte2 Computer obogcyte2 mutitsrak d connection Serial port D000 wi to Roboflow Power Switch Serial port to IN OUT external Liquid Handler GX 271 Connection to carrier Power Supply Compressed Air Inlet Compressed Air Outlet from pressure line to carrier 11 Roboocyte2 Manual Connecting the Carrier Plug the carrier connector into the according socket located on the Roboocyte2 s rear panel Connect the Compressed Air Outlet to the carrier Warning Confusion of the compressed air s inlet and outlet may destroy the device Have a close look at the preceding picture of the Roboocyte2 s rear panel and take care to connect the lines properly Connecting the Roboflow Plug the female connector into the male socket of the Roboflow Then plug the Roboflow connector into the socket on the Roboocyte2 s back Connecting the Roboocyte2 to the Pressure Line Connect one end of the provided compressed air hose ID 2 5 mm OD 4 mm to the water separator Connect the other end to the compressed air inlet on the Roboocyte2 s rear panel Connect the other provided compressed air hose ID 4 mm OD 6 mm to the provided water separator Make sure that the pressure line is closed Connect the free end of the provided compressed air hose ID 4 mm OD 6 mm to the pressure line
71. ion or baseline subtraction usually makes no sense for IV recordings 96 Analysis with Roboocyte2 Drift Correction The two cursors appearing in green color are used for correcting a linear drift of the holding current To do so current values at the two cursor positions are measured a straight line is calculated and subtracted from the original current trace To perform a drift correction you should move both cursors to a position representative for the linear drift typically but not necessarily at the beginning and end of the current trace or before and after the ligand application Then you can activate the drift correction by the check box Drift Correction and the drift will be more or less compensated Without Drift Correction With Drift Correction Recording 30 a e ESEESE Recording 30 a E mmm Y m Current n se a A AAAS eaa A a a a RE Pe 5000 10000 15000 20000 25000 30000 5000 10000 15000 20000 25000 30000 ms ms Now you can move the cursors around to get an optimal correction the current trace will be updated automatically according to the position of the cursors Baseline Subtraction In addition to drift correction a baseline subtraction often makes sense For example if you record ligand gated ion channels a baseline subtraction will cause a read out from the ROI which represents the ligand gated ion channel current unbiased by the leak current Taking the example s
72. ircumstances the use of any program feature will modify your original data Main features of the program are Review recordings Extract current or voltage values from your recorded traces Moving ROls regions of interest leak cursors and baseline cursors from positions defined in the recording script Performing P n leak subtraction This feature is accessible if the leak pulses have been recorded during the experiment Performing baseline and drift correction Generate IV dependencies Generate and fit dose response curves Exporting raw traces IV relationships and dose response curves with fit parameters in ASCII format 85 Roboocyte2 Manual Roboocyte2 Main Window ioi xi Fie Settings Help b a robo cyte2 44 Results Settings Info Liquids Injection O well ID Series ROI Minimum Pos Min Maximum Pos Max Average Area Left Right View Data List l Curve Dose Response Curve 0 Baseline Subtraction Drift Correction 0 Tl Leak Subtraction 0 Time Unit ms 0 Current n Current n Data Display Result Display 1000 2000 3000 4000 Time ms ch 9 100 4o Ha a 150 Voltage mi The main window of the Roboocyte2 software is divided in an upper section with the Tree View on the left side displaying the data saved in the file to be analyzed and the Data List on the right side In the lower left section the recorded data traces for the selected recordings are
73. l information about the composition of the liquids and the liquid handling are listed from left to right Well The well number for the recording B12 ID The recording ID from 30 to 36 ROI The number of the ROI Valve The valve number which has been transmitted from the script command Robo2 TransmitCompoundValve Rack The rack number transmitted by the script command Robo2 TransmitCompoundGilson Slot The slot number transmitted by the script command Robo2 TransmitCompoundGilson Tube The tube number transmitted by the script command Robo2 TransmitCompoundGilson Buffer The buffer assigned to the transmitted Tube or Valve Comp 1 The compound assigned to the transmitted Tube or Valve conc 1 The concentration of compound 1 unit 1 The unit of compound 1 94 Data List Injection tabbed Page Mame 1 Name of compound 1 Name of compound 1 Name of compound 1 Name of compound 1 Mame of compound 1 Mame of compound 1 Mame of compound 1 Name of compound 1 Name of compound 1 Name of compound 1 Name of compound 1 d Results Settings Info Liquids Injection m wal ID 1 a A 8 If Al 0 Compound ID 1 fw Al Compound ID 1 If Al 2 Compound ID 1 iv Al 3 Compound ID 1 iv AT 4 Compound ID 1 iv Al 5 Compound ID 1 Ww AT 6 Compound ID 1 If Al Compound ID 1 a 181 20 VoltageSteps TMCS i Al 32 Compound ID 1 Al 33 Compound ID 1 wo Al 34 Compound ID 1 Al 35 0 Compound ID
74. m a i 54 Another great help is that the Code Completion function also shows you the number and format of variables or parameters belonging to the respective command For example when you type Robo2 L the completion function offers you the command LeakCurrentCheck and informs you in a separate box about valid parameters In this case minValue maxValue numLoops and wait the format of witch is integer respectively TZ RecDisplay Clear 73 i Roboz 5et amplekate i 3AMPLERATE gt 74 Roboz L bai a a pubic LeakCurentCheck miri alue int masalae int numLoops int wait int bool PS de OARS Log leak current check PP LAA Max NUM_ROIS YG Robo2 Max _NUM_VALVES T3 ET MAXIMUM oO MASIMUM1 al for v A M4xIMUM2Z COount i Get MAXIMUM ae 3 a i 54 84 8 1 8 1 1 Analysis with Roboocyte2 The Roboocyte2 Analysis Software Analysis with Roboocyte2 The Roboocyte2 program is the offline analyzer part of the Roboocyte2 software package It is intended for analysis and review of the data for ASCII export and for visualization of the results The Roboocyte2 program uses both information stored in the database used for recording and information stored in the plate files Before loading a plate file the corresponding database has to be loaded When you close the program the database will be used to save the changes you made to your analysis Note Under no c
75. ms MCS GmbH and switch it on Please refer to the chapter Setting up the Roboflow The Roboflow works with 12 magnetic pinch valves and two peristaltic pumps termed Valve Pump and Waste Pump You can open and close the valves by a mouse click on the respective symbols Note You can open only one valve at a time Opening a second valve will automatically close the already open valve Start one of the peristaltic pumps with a mouse click on the On button The speed is controlled by the respective values defined in the Speed up down box The maximum speed for the valve pump is 10000 which corresponds roughly to 10 ml per minute Please choose the speed of the waste pump always higher than that of the valve pump to guarantee proper aspiration of the running solution from the well The maximum speed is 16000 which matches with 16 ml per minute The Clean Valves functions allow to clean rinse or prime the tubings in a comfortable way By pressing Start the Roboflow cycles from the First Valve to the Last Valve and opens the valves one after the other for the selected Rinse Time in seconds At the same time valve pump and waste pump are started with the selected speed values 57 Roboocyte2 Manual The Gilson Liquid Handler GX 271 Liquid Handler Tube Slat fa Rack 60 Tube D TFort Fort fi Valve 1 3 Valve 2 3 Perstaltic Purp Direction Foward C Backward If a Gilson Liquid Handler is con
76. n the Air Cushion button is selected the Slow Fast and Arrow buttons become active and you can move the carrier in x and y direction Air Pressure Resistance and Display Section Aur Pressure Check 3000 hPa O Resistance Check u Display C Welcome Screen C Generic Screen C Measurement Screen Click the Check button in the Air Pressure window to control the air pressure The result is displayed in hPa 1000 hPa 1 bar 14 5 PSI 750 Torr 29 5 in Hg Air Pressure Check 3000 hFa 49 Roboocyte2 Manual Click the Check button in the Resistance window to display the actual electrode resistances which will give back reasonable values only when the electrodes are immersed in an appropriate bath solution Resishance Check 475 EO hin U 123 EO hin In the Display section you can switch the Roboocyte2 display between the Welcome Screen and the Generic Screen and the Measurement Screen Display 0 Welcome Screen Generic Screen Measurement Screen Z Axis Section All functions in the Z Axis section refer to movements of the z axis Reference Home Park Pos Tube Oocyte Liquid Click the Reference button to start the reference movement of the z axis After performing a reference movement the z axis stops at a defined zero position Click the Home button to move the z axis to the home zero position Click the Park Pos button to move the
77. n the ROI where the maximum was detected in ms s or min Average The average current within the ROI Area The calculated area charge within the ROI in nA s The area is calculated from absolute current please note that this calculation gives meaningful values only after subtracting the baseline Left Position of the left ROI cursor Right Position of the right ROI cursor 92 Analysis with Roboocyte2 Data List Settings tabbed Page dij Results Settings Info Liquids Injection E well ID BaselneL BaselineR B Drift 1 Drift 2 Dr Basel L On Ls a A3 8 wl AS 16 0 145 O 30 o 527 0 wO A3 17 0 140 2 30 1 8 527 0 Ww A3 18 0 i 0 0 O 527 0 wo A3 19 0 140 O 30 1 e 528 0 wo a3 20 0 La e 30 5 we 527 0 E a 2 4290 4660 17100 8480 O 527 0 iw a3 22 0 Lao 30 7 8 527 T wooo a BY 0 140 0 30 o Ll 527 0 a l AS 4 VoltageSteps THICS wo A3 76 0 143 O J4 38 776 e 4 vw oA At 0 143 O 34 E E 596 P 4 wo A3 BB 0 143 O 34 5 O B16 G 4 wo aa y 0 143 O J4 8 O 536 Lal 4 The Settings tab includes the following information from left to right Well The well number for the recording B12 ID The recording ID from 30 to 36 BaselineLeft The position of the left baseline cursor BaselineRight The position of the right baseline cursor B Info whether baseline subtraction is active or not for the respective recording Drift 1 Position of the left drift cursor Drift 2 Position of the right d
78. nce NNT 1 Yoltage ps lt a er Wo Get 20 Current U E 2 0 A RST a 40 3 Amplifier Control i p 10044 I 1 cx 400004 0 60 Se a ee M AXC 0 1 2 a 4 5 Recording Freg 20000 Hz ie area a gt co es H I t sona Spoons f amv sf i2mv sh ae gt Start 2 Record Gain 1000 100 sampling rate 20 kHz Recording Clamp Set Holding Voltage Yoltage Current Ja n _set Amplifier Control P 1000 4 l 1 Cx 40000 4 W AXC Time s Recording Freq 20000 Hz Woa og es ME f a Sea Sh samy a g gt Stant 2 Record Gain 4000 100 sampling rate 20 kHz Current n Au aeo Recording Clamp Set Holding Voltage i i Yol ii Voltage A N aE i Li K Yi ree ny Th Das elo hata AeA A H pee E 5 e CUA rit cet a 0 a l 40 E Amplifier Control i E F 4000 I 100 CH 40000 4 0 0 W AE Recording Freq 20000 Hz Poa a es H A t sona eoa f eaw v sf ae gt Start 2 Record Time fa 31 4 1 Roboocyte2 Software Introduction Operate the Roboocyte2 robot collect and evaluate the data by using the Roboocyte2 and Roboocyte2 software The easy to use graphical user interface of the Roboocyte2 software makes daily work with the Roboocyte2 quick and easy Recording is started by a single mouse click The Roboocyte2 controls the run for all 96 oocytes automatically even including a wash cycle Thus the recording can go on overnight uns
79. ne ROI and Drift Cursors icon l to remove and display the drift cursors and the baseline cursors 98 Analysis with Roboocyte2 Result Display On the right side of the lower part you can choose in the Result Display whether the recording results are calculated as IV Curve or as Dose Response Curve Displaying and Fitting of Dose Response Data To use the feature of plotting and fitting dose response curves in the Result Display window two conditions have to be fulfilled already during recording the data Compounds and their respective concentrations have to be defined for the valves used and the respective liquid configuration has to be selected See chapter Linking Compound Applications to Recorded Data for details The recording script has to contain the two commands Command 1 Robo2 TransmitRecording REC_TAG_REF_COMPOUND or Robo2 TransmitRecording REC_TAG_ COMPOUND Command 2 Robo2 TransmitCompoundValve valve where valve is the variable for the valve number Robo2 TransmitRecording REC_TAG_REF_COMPOUND will cause the data to be plotted in the Dose Response tab of the Result Display but it will not be used for fitting This is for example useful for the application of a reference concentration to check the expression before starting the full dose response protocol Robo2 TransmitRecording REC_TAG_ COMPOUND will also cause the data to be plotted but the data will also be used for fitting a
80. nected and switched on the Liquid Handler part of the Liquid Handling tab page will become accessible Please refer to the chapter Setting up the Gilson Liquid Handler With the Gilson Liquid Handler section you can control all functions of the GX 271 separately Home brings all parts of the GX 271 to home position Reset has the same effect as switching the Gilson on and off Up brings the z axis of the Gilson to the most upward zero position The command Tube moves the z axis above the selected tube AND moves the z axis together with the tip probe if installed deeply down into the tube Warning If the wrong rack is selected the tip probe can be easily destroyed A Also be careful with your hands when you perform movements of the Gilson 58 Manual Mode ee Slot 2 Rack 24 Tube fi Selecting Racks and Tubes Tube Slot 3 Rack 60 Tube 1 TPort Port 11 OO amp 05 Bee Valve 1 Valve2 oo 4 Rinse Drain Valve 1 a Valve 2 a Select a certain rack from the Slot drop down menu and a Tube from the respective drop down menu Note There are five slots available racks will appear exactly as they have been selected in the actually active liquid configuration You have the choice between Rack Code 21 which holds 60 tubes 9 ml each or Rack Code 24 holding 8 tubes 20 ml each and Rack Code 60 with 4 bottles 180 0 ml glass or 250 0 ml polypropylene Testing the
81. ner of the Script window and the editor will open rL e j Edit C Dokumente und Einstellungen cramer Eigene Dateien Multi File Edit Search Script fe iff First Recordingscriptl js fff Copyright 2010 by Multi Channel Systems NCS C Robo Enab leRunt imeChecks true i RoboZ Logi Script To SCRIPT FILE pO om oo amp Pa for ivar i Of i lt Roboe SelectedWells cCount i4 10 H H war WellIndex Robhog SelectedWells il iz 13 current clamp mode 14 Roboz Log Current clamp at UO n 15 Roboz etHoldingturrent 0 16 Roboz SeccCurrente lamp iil Test Script Break Save Cancel Create I Protocol Test cript Output 82 Recording with Roboocyte2 Control Software Although you can use every text editor for reviewing editing or writing a script MCS recommends to use the built in script editor because it can test scripts for syntax errors as well as for some but not all logical errors Click on the Test Script button will execute the script and will tell you either that the script is ok or not Log Response O not in Range 100 10000 go to next oocyte Log Log Wait Log Recording protocol completed script finished OF seript aborted Error Function expected Line 107 Char 1 ControlbDisplay setyVaxisl o 30 If the Test Script function finds an error it displays the wrong expression and jumps to the respective line
82. nged 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 Requirements for the Installation The Roboocyte2 weighs more than 22 kg Always grip it tightly and do not carry it alone but with the aid of another person The movement of the well plate carrier can lead to vibrations of the workbench on which the Roboocyte2 is set up Therefore the Roboocyte2 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 Roboocyte2 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 it The external power supply is only for use with the Roboocyte2 Do not connect it to any other instrument 2 1 3 2 1 4 2 1 5 2 1 6 Roboocyte2 Manual Compressed Air Supply Even small amounts of water in the compressed air can lead to a corrosion of the carrier Other contaminations can also result in malfunctions Use only compressed air fulfilling
83. ns 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 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 3 1 Setting Up Hardware and Software Setting up Roboocyte2 Hardware and Software 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 confus
84. nsfer port 1 or 2 by using the respective buttons in the Liquid Handling tab of the Manual Mode Switch on the Minipuls 3 pump in backward direction by using the respective buttons in the Liquid Handling tab of the Manual Mode 67 7 1 Recording with Roboocyte2 Control Software Preparations for Recording General Preparations Either you plan to do recordings in manual mode or automated mode there are some preparations common to both Installing the Measuring Head Important The glass electrodes have to be backfilled with the electrolyte without any air bubbles Otherwise the electrode resistance will be too high The Ag AgCl electrodes have to be placed into the electrolyte but keep the blank silver part of the electrode outside the electrolyte to minimize slow voltage drifts over time Blank silver does not generate a stable junction potential Take a fresh measuring head out of the box be careful not to touch the tips of the glass electrodes Insert a fine pipette tip micro loader for example into the glass electrode as deeply as possible without breaking the delicate electrode tip and carefully fill about two thirds of the volume of the glass electrodes with the electrolyte solution so that the only Cl coated part of the Ag AgCl electrode can be inserted into the electrolyte slowly withdrawing the pipette tip during the process MCS recommends to use either 3 M KCI solution or a mixture with 1 5 M KAc 1 M KCI
85. og box Please click main menu Settings and select Options Liquid Handling M Gilson Connected Positions of TEWC probe Move to Liquid 2000 Lr Move to Docyte s00 um OF Cancel Activate the check box Gilson Connected and restart the software after switching the Gilson on During the software starts the GX 271 will be initiated and will perform its reference movements Important After changing the selection in the Gilson Connected check box you have to exit the Roboocyte2 program and start it again 65 6 3 1 Roboocyte2 Manual For often needed solutions like Ringer s solution or reference compounds it is recommended you use the transfer ports of the liquid handler To the transfer ports large bottles can be connected Please make sure to use the provided needle with the beveled tip if you plan to use the transfer ports of the Gilson GX 271 Otherwise the transfer ports may not work properly The Roboocyte2 supports two transfer ports The Gilson liquid handler is used for delivering compounds directly to the perfusion inlet of the TEVC probe The input flow is controlled by the Roboocyte2 via the Minipuls 3 peristaltic pump Multi Channel Systems MCS GmbH recommends to use a yellow tubing ID 1 42 mm see also Sources of Supply with the Gilson Minipuls 3 peristaltic pump and to use a pump speed of 25 rpm The resulting flow rate should be in the range of 5 to 6 ml per minut
86. onfigurations TestValves OF Cancel amp Settings For liquid handling X Directory for liguicds Eenmaa a E E a Ea EEE Channel EEE a TE a E E Configure Liquids with Valve Liquids Gilson Liquids Cancel Please see chapter Database Tool for dealing with the database 37 Roboocyte2 Manual Help Menu Fie Script Roboocyte Settings Tools g a n Open the Roboocyte2 Help menu to activate the online Help or the show the About dialog in which diagnostic hardware parameters are listed About Roboocyte2 roboecyte2 38 4 2 1 Roboocyte2 Software The Tool Bar i a Jir a Clicking on the icons in the tools bar gives you easy access to often used functions In addition you can access these functions in the menu ee Click on the Open icon to open a well plate generated with the Roboinject program gt Click on the Start icon to start a script controlled recording sequence Click on the Stop icon to stop a running script controlled recording sequence Click on the Alignment icon to open the Alignment Wizard For details please refer to the chapter Alignment please refer to the chapter Manual Mode Click on the Park icon to move the carrier into park position and the measuring head into the right dish of the two small dishes which can be placed in the metal rings at the backside of the carrier Lal D dap Click on the Manual Mo
87. or information on the installation of the rinse and transfer port station please refer to chapter Setting Up the Gilson GX 271 Liquid Handler 66 Compound Application GX 271 Rinse and transfer port station Rinse Drain Transfer port 1 Transter port 2 Important Please note that only the leftmost rinse drain and transfer ports can be used with the Gilson GX 271 liquid handler The other transfer ports have to be closed with plugs you can use convenient screws Do not remove the plugs otherwise the suction will not suffice to aspirate solution from the transfer ports Using the transfer ports The lines should be primed with Ringer s in the Liquid Handling Program before starting the recording protocol The transfer ports can also be used for applying larger volumes of solutions during the protocol Important In the script use the appropriate Gi son Valve1On or Gilson Valve20On commands to open the transfer port lines 1 or 2 respectively If the valve is closed liquid aspiration is not possible See the Roboocyte2 Scripting Language manual for more information Put the tube leading to transfer port 1 into reservoir 1 for example a bottle with frog Ringer and the tube leading to transfer port 2 into reservoir 2 Move the Gilson needle into the appropriate transfer port 1 or 2 by using the respective buttons in the Liquid Handling tab of the Manual Mode Open the solenoid valve of the appropriate tra
88. orrected data traces Baseline subtracted data traces Leak subtracted data traces All Parameters from the Data List Analyzed data from the Result Display such as dose response curves or IV dependencies All data will be exported as ASCII text either tab delimited or delimited by a user defined character The export function follows the principle WHAT YOU SEE IS WHAT YOU GET which means that you are exporting either the displayed traces or parameters extracted from the displayed traces Export Settings On the File menu click Export and select the appropriate settings in the Export Data Table to dialog box In order to export a data trace select the trace or a number of traces you want to export and open the export window Export Data Table to X EAD ocuments and SettingsrettingerEigene D ateienk Mult Channel Systems sAobooc Browse Available Columns Selected Columns Order BaselineCorrection Export Options Column Separator If Table l Trace f Tab Delimited Results F voltage Defined Character OF Cancel 107 Roboocyte2 Manual Browse through the folders to define the path for the export file Switch from the window Available Columns the desired columns to the window Selected Columns by clicking the arrow buttons between both windows To change the order of the selected options please select the respective option which will be highlighted in blue and use the arrow
89. ors drift cursors in green baseline cursors in blue and ROI cursors in red will be displayed at positions which have been defined in the corresponding recording script Zooming and Rezooming i When you press this button the mouse pointer will change its appearance and you can specify a rectangular region in the data display to which you can zoom Use the Reset button to get back to the original view Exporting or Printing of the Display When you click on the Print icon i additional icons appear a7 i by which allow you to save or print out the display You can from left to right copy the display figure to the clipboard save the display in all kind of formats jpeg tiff png etc print the display or have a preview before using these functions 106 8 4 Analysis with Roboocyte2 Data Export to ASCII Format The raw data recorded with Roboocyte2 software is saved in the plate rpf file You can review and evaluate the raw data offline with the Roboocyte2 software However if you like to study the data in more detail or to print a custom plot you can export the raw data in ASCII file format The extension for the file name of the output files is dat Some programs allow to open ASCII files directly others have filters for importing ASCII For example you can import the data into Origin or Microsoft Excel Use the export function of the Roboocyte2 program for Raw data traces Drift c
90. our buffer s and compound s To delete the complete configuration or the configuration of the selected valve please use the buttons Clear Complete Configuration and Clear Configuration of Selected Valve Use Exit if the compositions are Okay Defining a Buffer Click on the icon to open the buffer editor Edit Buffer Edit Buffer rid 4 fi offi1 gt bilo X id ild 4 i f5 gt gt X H Concentration Comment Compound Nall C KCI 2 1 Buffer ame Comment Calle HEPES OK Cancel In the Edit Buffer dialog you have to define the buffer in the left half of the dialog Specify the Buffer Name and the pH value of the buffer and write a Comment if necessary By clicking on a buffer name in the first column you can define the compound composition of the buffer and the concentration in the right half of the dialog Generate new rows for additional entries by clicking the Add Raw icon i and delete rows with x l 62 Compound Application Defining a Compound Click on the compound icon r to open the compound name editor Edit Compound H 4 4 f b bl oe x H Compoundh ame Comment GABE Please define in this dialog compound names with additional comments for example GABA ACh or ATP Save the definitions via Save button El Click the OK button to confirm the settings The definitions of buffer and or compounds are sent to the dat
91. out the expected noise levels Gain 1000 100 sampling rate 20 kHz Current n Da Ree Ren A N SRG Pee A ee 4 ss 2 t 60nd afena Sf Ea sf 12mv Ala F Gain 1000 100 sampling rate 10 kHz aap E z 20 5 ee 2 40 a GO ea a Ee Boa GSE SS 2S SE SY SS T 0 1 2 3 4 a Time gt Gain 1000 100 sampling rate 1 kHz a ss MIF g na ea Sf tam e sy a 0 20 F 0 a 40 All 0 1 2 3 4 F Sacre rere er Ra Re Eme Rae Nee ne ern a A oa iss ME t na eona Sf tani Sy 2m ey 30 Au abeyo A aw abeo Au 5e Recording Clamp f Voltage C Curent Set Holding Voltage TE mi Set Amplifier Control p 1000 100 4 LA oj IY AYE Recording Freg 20000 Hz p Start Record Recording Clamp f Voltage C Curent Set Holding Voltage TE m Set Amplifier Control e 0o io e os IY AXE Recording Freg 5000 Hz p Start Record Recording Clamp f Voltage C Current Set Holding Voltage TE mi Set Amplifier Control ejo o a os IY AXE Recording Freg 7000 Hz p Start Record Setting Up Hardware and Software Decreasing the sampling rate will decrease the current noise level increasing the proportional gain will increase the current noise at a given sampling rate Gain 100 100 sampling rate 20 kHz Recording Clamp Set Holding Voltage SL pear es tsa RI I tana ta
92. p of the GX 271 Liquid Handler Unpack and set up the Minipuls peristaltic pump MP3 and the Gilson GX 271 liquid handler according to the descriptions in the Minipuls 3 Peristaltic Pump User s Guide and the GX 271 Liquid Handlers User s Guide from Gilson in the immediate vicinity of the Roboocyte2 preferably on the right side next to the recording z arm Do not place the bath reservoir or the waste bottle too near to any AC voltage sources for example the liquid handler itself or the Minipuls pump Electrostatic interference can be introduced by the voltage source and travel along the perfusion line resulting in a high noise level Multi Channel Systems recommends that the Minipuls 3 pump and the Gilson liquid handler are placed on the right hand side next to the Roboocyte2 please see the figure Setup Gilson Liquid Handler to minimize the travel distance of the solvent Make sure the rinse transfer port station was installed to the locator pan Place the locator pan onto the locator plate The rinse transfer port station should be on the top right position The four metal noses of the locator plate should fit into the holes of the locator pane Set up the z arm and guide foot according to the chapters Z Arm Setup of the GX 271 Liquid Handlers User s Guide from Gilson You will not need the Fraction Collection Valve Installation Adjusting the Z Arm Height of the GX 271 Liquid Handler After z arm installation according to the GX 271
93. proportional gain value determines the speed of voltage clamp whereas the integral parameter determines how fast the remaining voltage error is compensated Please refer to chapter TEVC Recording Background for details Click the button Save and Accept under the IV protocol display to save the present IV protocol and to start the execution of the protocol Click Cancel to stop it 59 5 4 Roboocyte2 Manual Manual Mode Liquid Handling The Liquid Handling tab in Manual Mode gives you access to all functions of the Roboflow valves and pumps and the Gilson Liquid Handler GX271 if connected w Manual Mode Movement Measurement Liquid Handling Robo Flow Gilson Valves Liquid Handler s 3 3 3 3 8 we e ne fo alal 3 3 3 s al all ia all a all Clean Valves First Y alve fi Rinze Time z 2 Penia Pume Direction Last Yale 12 Forward Stat Stop Backward The Liquid Handling tab page is divided in two windows On the left side you have manual control over the Roboflow System and on the right side you manipulate the Gilson GX 271 by hand 56 Manual Mode Roboflow System Valves alal alel 3 3 alal al 8 a 3 Valve Pump Waste Pump Un Un Speed Speed 1000 1000 Clean Valves First Valve fi Rinse Time a 2 12 iz Last Valve Start 2 Stop Connect the Roboflow System from Multi Channel Syste
94. quickly find out what they do Important again DO NOT press the Set Alignment button This button should only be pressed with installed measuring head aligned to the surface of the alignment tool 24 Setting Up Hardware and Software 3 5 2 Testing the Roboflow Valves and Pumps The Roboflow was designed to work with the Roboocyte2 and is therefore completely controlled by the Roboocyte2 software For an initial test open the Liquid Handling tab of the Manual Mode window Manual Mode Movement Measurement Liquid Handling Roba Flow Gilson No Gilson liquid configuration loaded Valves Liquid Handler a 3 2 3 a 1 e ed Tube Slot Tube Valve Pump Waste Pump On On iz Port Speed Speed 10 1000 Clean Valves First Valve fi Rinse Time 2 Last Valve 12 Foward Stat 2 Stop f Backward Testing the Pinch Valves Press the valve buttons one after the other from 1 to 12 and check whether the respective valves open Testing the Peristaltic Pumps Press the Valve Pump button and check whether the pump located on the movable slide runs Then press the Waste Pump button and check whether the pump on the upper right side of the Roboflow front panel runs The number of the pump Speed is roughly equivalent to ul min the maximum speed for pumps is 10000 and 20000 for the valve pump and waste pump respectively Overall Test You can also use
95. rdingly The corresponding display will be scaled to the specified values for current and voltage 0 z 100 lt or z 200 T 3 300 50 x 400 0 2 4 G 10 Time fa gt a og ws A E t Ana ioa o o et aoe Additionally you can change the scaling by using the arrow buttons 41 Roboocyte2 Manual Using the mouse for scaling Instead of entering numbers to scale the axis you can use the mouse to move or scale axes Move the mouse pointer above the respective axis the pointer will now appear as a hand Hold down the left mouse button to grab and move the axis When you hold at the same time the control key keyboard the mouse pointer will change to an arrow and moving the mouse or mouse wheel will re scale the axis For tracking the axis manually click on the axis and the mouse pointer will change into a hand To scroll the axes please move the mouse with the hand pointer For zooming the axes press the CTRL key and keep it pressed while moving the mouse or the wheel of the mouse The mouse pointer turns into a double arrow symbol Automatic scaling Automatic scaling means that the display will change its scaling depending on the amplitude of voltage or current When you push one or both auto scale buttons the auto scaling mode is activated Pushing the buttons again deactivates the auto scaling Zooming and resetting When you press this button the mouse pointer will change its appearance and you can spe
96. rift cursor Dr Info whether drift correction is active or not for the respective recording Baseline Average Average current between BaselineLeft and BaselineRight Lon Info whether leak subtraction is active or not for the respective recording Ls Info about the type of leak subtraction protocol used 0 none 2 P 2 4 P 4 93 Roboocyte2 Manual Data List Liquids tabbed Page Ful Liquids Injection Valve Rack Slot Tube Mm om FF ws M E 1 _ el dij Results Settings Info i Well ID a _ Al 8 AT 0 0 w A 0 GEJ 2z D TEY a 0 w AT 4 0 wf Al 5 O wt A 6B D AT 7 0 a 181 4 YoktageSteps_TMC3 BEY 3 amp 6 Biel 47 0 Al 46 D Al a el l Buffer ORI empty empty empty empty empty empty empty ORI ORI ORI ORI Comp 1 empty compe compe compe compe compe compe compe empty empty empty empty CONE 100 ee l l l unit 1 nb rib nti nbi nbi rit nti nhi rib nti nii nbi Comp 2 empty empty empty empty empty empty empty empty empty empty empty empty CONC Se ee ee eS D D A unit Z mbl mbi mb mbl mbl mbi mb mbl mbi mb mbl mbl Comp 3 empty empty empty empty empty empty empty empty empty empty empty empty CONC D ee eae ea eaa j aaa SS ee ee unit 3 mtl rti mbd mt mtl mti mt m mti mtd mt mtl In the Liquids tab al
97. rk server from a second computer for offline analysis you have to manually relink the original database from the second computer after the program installation A new database will be set up during program installation that you will not need Please see chapter Linking to the Database for more information Otherwise it is possible that the installed software does not work properly Double click Setup exe on the installation volume The installation assistant will show up and guide you through the installation procedure iz Roboocyte 1 0 0 InstallShield Wizard multichannel Welcome to the InstallShield Wizard for mS Roboocyte 1 0 0 innovations in Bectrophysiology The InstallShield R Wizard will install Roboocyte 1 0 0 on vour computer To continue click Next Back Cancel Follow the instructions of the installation assistant until the installation is complete i Roboocyte 1 0 0 InstallShield Wizard multichannel InstallShield Wizard Completed The InstallShield Wizard has successfully installed Roboocyte 1 0 0 Click Finish to exit the wizard Back Gancel The Roboocyte2 software and the Microsoft Access database file accdb are installed on your computer 23 3 5 3 5 1 A Roboocyte2 Manual Testing the Roboocyte2 Each delivered Roboocyte2 System has undergone extensive tests at the MCS site Please follow the provided instructions to perform initial tests just to make sure that every
98. s over the x y table please remove it as soon as possible in order to prevent a corrosion of the carrier Handling of the Z Axes Do not move the z axes by hand Breakage may occur Always use the software controls to move an axis up and down Change the probe with great care Stabilize the z axis with one hand while changing the probe with the other 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 z axes 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 Handling of the Ready to Use TEVC Probes The chloride coated silver wire is sensitive to light Always keep it dark 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 z axis If you want to plug the silver wire directly into the sockets of the z axis use only 0 4 mm silver wire for the electrodes A wire with a greater diameter will damage the connectors of the z axis irreversibly Important Safety Advices 2 1 7 Regular Backups 2 2 2 3 You or the administrator
99. sMulti Channel SustemssAoboocytes 201 2 Nov 15 acedb Click on Options to open the options dialog LS Sti x Liquid Handling W Gilson Connected Positions of TEWC probe Move to Liquid 2000 uri Move to Docyte s00 um OF Cancel This window allows you to select whether a Gilson liquid handler is connected or not In addition you can define here the liquid position i e how deep the measuring head should move into the well before performing resistance measurement or electrode voltage offset correction Finally you can define here the position of the initial movement towards the oocyte to start the impalement process The default value of 800 um means that the electrode tips move to a position 800 um above the bottom of the well If the oocyte would have a diameter of 1 2 mm it would mean that the glass electrodes would be moved 400 um deep into the oocyte 36 Roboocyte2 Software Tools Menu File Script Roboocyte Settings Manual Mode Select Liquid Configuration Database Tool Define Liquid Configurations In Tools menu you can open the manual mode window select an existing or define a new liquid configuration and activate the Database Tool Concerning the Manual Mode please read the respective chapter For details concerning the liquid handling please refer to the chapter Liquid Configuration and Liquid Handling Select Liquid Configuration Available Fluid Control C
100. script Liquid Handling Preparing Solutions Before starting an experiment you should prepare all solutions to be used and prime all tubings of either Roboflow or Gilson Liquid Handler You should do this after installing the measuring head and after connecting the inflow and outflow tubing to the measuring head Roboflow In order to prime the tubings open in main menu Tools the Liquid Handling tab of the Manual Mode window Connect all 12 tubings via the magnetic valves to the 12 fold manifold and place the tubes into the desired solutions If you want to use less than 12 different solutions in your experiments place the tubings not used in buffer solution because the solution exchange systems works properly only when all tubes are filled with liquid Valves 2 3 4 5 G a a a a a a f a a 10 11 12 a a a a a a Valve Pump Waste Pump On On S peed Speed 1000 1000 Clean Valves First Valve fi Rinse Time 2 Last Valve 12 Stat 2 Stop To start the priming procedure be sure that all tube endings are placed in the respective solutions Either put a small dish underneath the measuring head or use one of the small dishes either on Park or Resistance Position Please use the respective movement commands in the Movement tab of the Manual Mode Select 5000 for speed of valve an waste pump and 20 s for the Rinse Time Then press Start and all 12 valves will be opened sequen
101. single voltage steps should be executed with a non recorded waiting time in between the function Wait Time between Sweeps can be used The potential during the wait time between sweeps can be chosen with the Potential for Wait Time function The values in the boxes for Baseline and ROI Region Of Interest define the left and right boundaries for determination of baseline and current respectively Choosing the correct boundaries for the baseline current calculation is important for the correct calculation of leak subtracted current if leak subtraction is enabled P N Leak Subtraction Leak Subtraction MW Enable Leak Sweeps E Leak Holding mE mi If you enable the Leak Subtraction check box the parameters for defining the P N leak subtraction will be accessible and leak subtraction will be performed when the IV recording is executed The leak subtraction procedure follows the known standards for P N leak subtraction You can choose between 2 and 4 leak pulses the Leak Holding potential from which the leak pulses are started and whether the polarity Type is Standard Reversed or Alternate Amplifier Control Parameters F Gan Garm Sampling A ate 3000 4 100 20000 Hz The built in digital amplifier of the Roboocyte uses a PI control loop feedback mechanism where P stands for Proportional and I for Integral Units for P and I are nA and mV and 1 s respectively Generally spoken the
102. slits Proceed in this manner for all twelve valves If you are going to use only some of the tubes please connect the useless tubes to Ringer solution for example but do not leave single manifold connections open 14 Setting Up Hardware and Software Attaching the Tubing to the Valve Pump Open the screw of the press bracket of the valve pump and remove the press bracket Insert the tube section PPRT1 14 provided for the valve pump 6 Remount the press bracket and close the screw carefully Please start the valve pump by software control to move the tube section into the pump housing until the spacer stops this process 15 Roboocyte2 Manual Software Control for Inserting the Tube Section PPRT1 14 in the Valve Pump Please switch to the Manual Mode in Roboocyte2 control software Click the icon a or select main menu Tools Manual Mode Open the Liquid Handling tab page Manual Mode Movement Measurement Liquid Handling Roba Flow Valves aia eee aes Valve Pump Waste Pump Speed Speed 100 100 Clean Valves First Valve fi Rinze Time a 2 Last Valve 12 Stat Stop Press the button Valve Pump to run the pump as long as it needs to insert the tube section into the pump housing until the spacer stops this process Stop the pump by clicking the button again Adjust the orientation of the manifold for connecting the short part of tube section to the outlet noz
103. thing is alright and that no damage has occurred during shipment Testing the Movement of Carrier and Z Axis On the main menu Tools click Manual Mode or click the icon F The manual mode dialog box opens with the Movement tab page active Warning DO NOT press the Set Alignment button This button should only be touched with installed measuring head aligned to the surface of the alignment tool Manual Mode l X Movement Measurement Liquid Handling Global Air Pressure Reference Home heck 2000 hPa Coarse Position Set Alignment Resistance Change Flate i 5km u 123 kOhm 2 AMIS Plate Carrier Plate Greiner Standard 128 Reference Home Park Pos Reference Home Park Pos Tube Tube Docyte Liquid well fa Ar Cushion Opm a Y 150000 pm Eai Fast The Movement tab is divided into different sections Global Plate Carrier Air Pressure Resistance and Z Axis For a detailed description of all functions please refer to chapter Manual Mode Testing the Air Pressure Press the Check button in the Air Pressure section The actual air pressure will be displayed Testing the Reference Movement Press the Reference button in the Global section The z axis and the well plate carrier should perform their reference movements Getting familiar with the Movement Tab Page Try the other buttons of the Movement tab window you will
104. tially for 20 s each If there are still air bubbles in one or several tubings repeat the rinse procedure until all air bubbles have gone The settings about 1 7 ml for every solution will be used for each rinse run 75 Roboocyte2 Manual Gilson Liquid Handler When using the Gilson Liquid Handler the manifold will not be used and the tubing coming from the Gilson peristaltic pump is directly connected to the inflow of the measuring head Therefore this tubing will be the only one that has to be primed before use Similar to the prime procedure when using the Roboflow you can use again the Liquid Handling tab of the Manual Mode window In order to work with the Gilson liquid handler be sure that you have selected Gilson Connected in the Options of the main menu Settings Liquid Handling If Gilson Connected Positions of TEWC probe Mowe to Liquid 2000 um Move to Docyte s00 um OF Cancel In addition you have to select a Liquid Configuration for the Gilson in main menu Tools Define Liquid Configurations Please use the predefined liquid configuration Prime for priming from one of the two transfer ports T Ports Liquid Handler Tube Slot E Rack 60 Tube D Peristaltic Pump Direction Forward Backward Put the transfer port tubing into an appropriate solution move the Gilson probe needle to transfer port 1 or 2 open valve 1 or 2 and switch on the Gilson pump in
105. ual mode click button Check in the Resistance window Resistance Check 475 kOhm U 123 kOhm The electrode resistances for current and voltage electrode are displayed in the two small windows beside Both impedances should be approximately 500 kilohms Performing Voltage Clamp with the TMC You can run a test recording with the test model cell in order to check the speed and accuracy of the voltage clamp either in continuous recording mode or by a applying a voltage step protocol 26 Setting Up Hardware and Software Continuous Recording Switch to the Measuring tab page of the Manual Mode window Manual Mode Movement Measurement Liquid Handling Move Carrier ao to Well Jaq Home Move into Liguid Liguid DE eet Clear Offset Resistance Check n a Valve Pump all 1000 Waste Pump all 1000 Recording Clamp Set z Voltage nv Set f Current H Control 1000 a 9000 a Set as Default Recording Freq 20 Hz gt Start Stop Record Voltage Step Protocol Load E dit Create Protocal lt __ _ _ Current n Alu S660 A Time fal if 105 3 too S10 a 0 S20 w amp Current n 0 1000 2000 S000 Time ma A y f 10 S10 Sm E a Apply Stop Start the recording in the Current Clamp mode by clicking on the Start button Wait a few seconds and press DC Offset to compensate potential
106. ult Display there are a number of icons changing the appearance of the plot Data points only ae ___ Data points and lines E Data points and least square fits sgn ___ Open and close the dose response fit section 101 Roboocyte2 Manual Result Display Dose Response Curve Click on the Try to fit a Logistic Curve on the Data Points icon Logistic Fit 4P section of the Result Display to open the dose response W Cumwe Dose Aesponse Lurve b Logistic Fit 4F 1000 Lower Asymptote mir gt on 13080 5 n Sane Upper Asymptote ymax J476 nA 4000 ECSO ICSO F000 00956 mbl E000 F Slope S teepness 5 1 45 7 7000 8000 Calculate Fit ary Par 3000 Estimate Start Parameters 10000 41000 A mo 0 01 01 j Show Start Parameters ATF Concentration rot er Calculate Fit Start the curve fitting by a click on the button _Caloulate Fit The start parameters for the fit are calculated automatically If the fit does not converge you can try to type in parameters manually MW Cumwe Uose Response Curve Logistic Fit 4F 1000 Lower Asymptote y _min 2000 11755 n a Upper Asyroiptote ymax 256 1 n 4000 ECS0 IC50 5000 00369 mbi z Ennn F Slope Steepness 1 46 7000 8000 Calculate Fit ary Par 3000 Estimate Start Parameters 10000 41000 p mo BIDR 0 01 01 j Show Start Parameters ATP Concentration mb
107. ume of a well ensures that a flow rate of about 4 ml min achieves a rapid and efficient solution exchange with effective compound usage You can easily implement your own automated drug saving strategies into your experimental setup The movable peristaltic pump is connected to the measuring head inlet and is for compound application The peristaltic pump fixed on the upper right side of the front panel of the Roboflow device is for aspiration to the waste bottle The endings of the perfusion inlet and outlet tubings are fixed to the measuring head at different heights which keeps the fluid level constant You can either control the opening of the valves as well as the speed of application manually during manual recording or use the designated commands in a script For details refer to the Scripting Language Manual The definition of compounds and buffer for the Roboflow System is in principle the same as in other liquid handlers It is described in the next chapter Using the Gilson Liquid Handler Using the Gilson Liquid Handler The liquid handling of the Roboocyte2 System is possible either with the Roboflow System from Multi Channel Systems MCS GmbH or with the liquid handler GX271 from Gilson To connect one of these systems please read chapter Setting up the Roboflow System and or Setting up the Gilson GX 271 If the Gilson Liquid Handler is used you have to activate the communication with the GX 271 in the Options dial
108. upervised The large amount of data generated by the Roboocyte2 can be managed with a Microsoft Access 2010 database not included Multiple users can access the database over a network The high degree of automation and flexibility for such a demanding task is made possible by using scripts small text files containing commands Users can write their own scripts with any basic text editor or with the editor embedded in the Roboocyte2 program All experimental settings are defined within the script You can write a script for any kind of experimental setup Once the appropriate script is loaded into the Roboocyte2 software simply click the Run button to start the robot The script is then performed without the need for further customization and supervision The Roboocyte scripting is based on Java Script with many commands specifically designed for the Roboocyte2 It allows the full automation of various applications For further information on the Java Scripting Language please refer to the separate Java Scripting Language manual Although the main use of the Roboocyte2 will be automated recording there is a Manual Mode option with which recordings can be performed on single oocytes in a non automatic fashion Although all functions will be still controlled by the Roboocyte2 software single steps such as impalement clamping the cell performing solution changes etc are under real time control by the user 33 4 2 Roboocyte
109. user s manual you need to adjust the z arm height to the appropriate needle length Please see also chapter Adjusting the z travel height in the GX 271 user s manual 19 Roboocyte2 Manual Set the z arm to the height of 175 mm by using the ruler integrated into the z arm marked by a blue arrow in the preceding illustration Insert the stop pin into the hole labeled S2 marked by a blue arrow in the preceding illustration Installing the Probe and Transfer Tubing Install the provided needle to the GX 271 as described in the chapter Probe Installation of the GX 271 Liquid Handlers User s Guide from Gilson Install an appropriate pump tubing to the Minipuls 3 pump as instructed in the Minipuls 3 Peristaltic Pump User s Guide from Gilson Multi Channel Systems recommends the use of a Tygon pump tubing yellow yellow see Sources of Supply for details Screw the FEP tubing assembly onto the probe holder of the GX 271 Connect the other end of the tubing to the pump tubing by using elastic tubing adapter 200 51 and a 200 16 coupling Connect the other end of the pump tubing to the Roboocyte2 measuring head via the provided tubing assembly Setup the Gilson Liquid Handler GX 271 Connect the pump tubing to the Connect the tubing of the perfusion inlet of the measuring Gilson liquid handler GX 271 head of the Roboocyte z to the MP3 pump tubing Gilson Liquid Handler Roboocyte Minipuls 3 Pump This schematic illustration
110. ve arrow keys Alternatively you may use the according buttons on the keyboard Page up and page down keys for the z axis and the four arrow key for the carrier Fine tune the position until the alignment device is almost touched by the tip of the electrodes If you do this the first time you may have difficulties seeing the TEVC probe in three dimensions It may appear to be centered correctly but instead the probe is still a way above the alignment device and shifted to the front It is very important that the capillaries are centered correctly on the cross hair but the height of the tips is not so important It is recommended to lower the TEVC probe as low as possible without touching the surface of the alignment device because the glass electrodes are not so flexible and thus tend to break easily A stepwise movement is part of the automated recording sequence and achieves a gentle and reliable impalement with both electrodes 73 7 3 Roboocyte2 Manual If you need to start the alignment again for example if you have moved the carrier accidentally by hand click Cancel and restart the alignment process from the beginning Adjustment position recording Tips of the microcapillaries Adjustment device Click Next The current position is saved and the old alignment position is overwritten All following movements will be related to the new position now Warning Do not click Next if the alignment has not been finished
111. xperiments manually e To test the quality of oocytes before starting automated script controlled experiments e To test the expression before going automatic e To test new IV protocols or solution exchange sequences before implementing them into a script e To find an optimal impalement procedure for a given batch of oocytes Move Carer Go to Well Jat Home Move into Liquid Resistance Liquid Docyte Impalement Up t 4 _Doote ZUb40 urr 13 20 30 40 50 69 Pump 2 51 Roboocyte2 Manual The upper section consists of command buttons for movements amplifier controls before impalement and valve and pump control buttons Use Go to Well to move the carrier to the selected well and Home to move it back to home position Clicking the button Liquid moves the z axis down to a well for example into the solution of the well Clicking the button DC Offset performs a voltage offset compensation on both electrodes and is active only when a recording in Current Clamp mode is started Click the button Clear Offset to cancel the offset compensation This is useful to see the real electrode offset of both electrodes Click the Z Up button to bring the z axis back to the zero position Click Oocyte button to move the z axis for example the electrodes down to impalement position by default 800 um above the well plate bottom If the initial impalement movement Oocyte is not sufficient to
112. y default or in the specified plate folder Entries in blue are well plates stored in the Roboocyte Plate folder after creating them with the Roboocyte2 program itself Finally entries in black designate plates found in the database but not in the respective folders This can happen when you moved plate files to other folders or when plate files have been deleted After loading the well plate the well plate section will look like the following screenshot if all oocytes on the plate have been injected SOOCOOOOOOOOCO SOOOOOCOCOOOCOCO OOOO SOSOOOOOOOO To select a well for recording click on the respective well You can also select rows or columns by clicking on the respective row letter or column number and you can select the whole well plate by clicking on the MCS logo gt Selected wells will be marked with a red triangle like this ical It is not possible to select well H12 because it is used for the alignment tool Display Control Functions You can change scaling and appearance of the data display windows by using either keyboard or mouse All scaling and display functions can be controlled by script commands as well Please refer to the Scripting manual for details Changing the scaling with keyboard inputs or mouse Below each data display you will find a bar showing the actual settings of the respective data display If you change these values by keyboard input or mouse click the scaling of the data display will change acco
113. zle of the manifold Please see the picture above 16 Setting Up Hardware and Software Attaching the Tubing of the Waste Pump Connect the tubing to the provided connectors of the waste pump PPRT2 29 The silicone tube ID 1 0 mm OD 2 4 mm length 540 mm coming from the left side of the aspiration pump is directly connected to the outlet of the measuring head of the Roboocyte2 Please guide this tube also through the plastic tube holder to ensure the correct attachment The PVC tube ID 2 0 mm OD 4 0 mm length 1 m coming from the right side of the aspiration pump leads to the waste bottle Outlet Connect the tube section from the valve pump to the inlet connector of the measuring head of the Roboocyte2 This is the upper one of both provided connections Connect the suction tube coming from the waste pump to the outlet of the measuring head which is the lower connection of the measuring head below the inlet Please see the picture 17 3 3 Roboocyte2 Manual Setting Up the Gilson GX 271 Liquid Handler For high throughput screens the Roboocyte2 can be used with an industry standard liquid handler from Gilson optional the GX 271 Warning Only hardware configurations and probes provided by Multi Channel Systems GmbH are supported Other versions may lead to incompatibilities up to damages to the hardware needle breakage for example Please verify the system configuration with Multi Channel Systems GmbH if you obta

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