User Manual
Contents
1. Zero 3 F2 Zero Zero 4 Chapter 3 15 16 the display will automatically return to the force menu Re enter the zero menu using the arrow down button choose the next transducer with the F1 button and zero with F2 Repeat this procedure until all transducers have been zeroed To simultaneously zero all channels enter the zero menu and press the right arrow and F2 at same time Note there should be no force applied to the transducers when zeroing Heat menu In this menu the heating and temperature settings are controlled The F1 button turns the heat ON and F2 switches it OFF The temperature the myograph heats to Set temp is decreased with the button and increased with Act Temp is the actual temperature measured by the external temperature probe when connected to the interface If there is no probe connected Act Temp OFF will be displayed Note Whenever the system is turned on by the power switch on the rear panel the heating is automatically off until turned on by the user When the heating is on the set temperature will be reached in 20 minutes Recorder menu The 610M Myo Interface has four 1 V full scale analog outputs which provide the force signal in volts for connection to an external data acquisition system By default the interface is set at 20 mN where 20 mN corresponds to 1 V As the force value will vary according to the maximum response from the preparation used the2. optiona 0000 n 5 5 5 Geeet 9600000000600000900000000090090900090090000900909000000900009000090090000090000000000000000090000090000000000090000090000000000009000000000906 h RS serial connection Suction bottle optional Figure 2 1 The complete Tissue bath system Model OOMO The dotted lines represent op tional connections to the system 2 2 Setting up step by step The chapter contains a complete step by step description of how to set up a complete Tissue Bath system as illustrated in fig 2 1 1 Myo Interface PC connection Data acquisition is possible either by connecting the Myo Interface directly to a PC or through a PowerLab data acquisition and analysis system optional I Direct PC connection Connect the Myo Interface to one of the COM ports on the PC using a serial cable Cable not included PowerLab optional Follow the DMT Quick guide instructions delivered with the PowerLab sys tem to install the PowerLab driver and LabChart software on the computer Follow the instructions provided in the DMT QuickStart guide Data acquisi tion Tissue bath system 7OOMO and PowerLab LabChart User Manual 700MO Chapter 2 13 14 2 Gas supply The gas supply into the chamber is a plastic tube which is conveniently attached to the stainless steel suction pipe The tube must be placed in the chamber in order to have the solution aerated described below Connect the main gas sup ply to
3. 11 PESE Neige IRR NEN 12 Chapter 2 Setting up T T T 13 2 1 The complete Tissue bath system 13 WD SIOD DI UO uu uman a 13 2 3 The first weight calibration 14 Chapter 3 The Myo Interface 15 3 1 Turning on the Myo Interface 15 3 2 Main menus and submenus 15 Chapter 4 The Tissue bath unitl 19 4 1 Change and adjustment of mounting supports 19 4 2 Force transducer calibration 20 4 3 Checking force transducer 22 4 4 Force transducer replacement U 23 9156 u xu uuu E R 24 Chapter 5 Getting started T T nnan 21 eM 2 UO RTT 21 5 12918 EE
4. MEI EMEN ME MuR ME e Function keys 1 2 Myo Interface rear panel Gas regulator needle valve T A 1 Gas input T P L L Power connector Suction RS 485 port for serial connection to PC 4 Recorder outputs e e RS 282 port for serial connection to PC Temperature probe User manual 700MO Chapter 1 11 12 1 3 Tissue bath unit Micropositioner e Myograph pin support connected to micrometer DMT Tissue bath system Model 7OOMO port connected to transducer Force transducer pin Chapter 2 Setting up 2 1 The complete Tissue bath system Myo Interface front panel Power supply PowerLab data acquisition system optional oooooo j DMT CS200 Pulse Train Stimulator optional PC data acquisition and analysis software optional Power supply Connection to oxygen supply BNC Cables eee ee eee eee ee eee eee
5. If the endothelium is undamaged by the dissection and mounting procedures then a substantial relaxation will occur With complete removal or damaged endothelium a partial relaxation or no relaxation to acetylcholine is observed It is important to note that the amount of NO or EDRF in a vessel is often dependent upon its size In certain vessels endothelium derived hyperpolarising factor EDHF can contribute more or less than EDRF and in other vessels the same stimulation with ACh can promote release of endothelium derived contracting factor EDCF Therefore it is important to check the existing literature in order to determine the expected response in your particular vessel with the given concentration of agonist Procedure for checking endothelium function Step 1 PSS 3 uM NA Stimulate for 3 minutes Step 2 Add 10 uM ACh Wait 2 minutes Step Wash out 4 x with PSS Ready for experiment User manual 700MO Chapter 5 29 30 5 4 In Vitro Experiment 1 Noradrenaline contractile response The purpose of the present protocol is to determine the sensitivity of rat mesenteric small arteries to the vasoconstrictor noradrenaline norepinephrine with a cumulative concentration response curve Background Noradrenaline norepinephrine causes contraction of mesenteric small arteries through activation of o adrenoceptors whereas noradrenaline activation of B adrenoceptors causes vasodilatation As the purpose
6. 1096 50 60Hz User manual 700MO Appendix 9 45 46 Notes DMT Tissue bath system Model 00MO DANISA M10 T C k COL DMT A S Skejbyparken 152 DK 8200 Aarhus N Denmark Tel 45 87 41 11 00 Fax 45 87 41 11 01 www dmt dk sales dmt dk support amp dmt dk DMT Asia Everwin Gardens Rm 502 Block B 521 Wanping Nan Lu Shanghai 200030 China Tel 86 21 64869685 Fax 86 0 21 64280591 www dmt asia com sales dmt asia com Support dmt asia com DMT USA Inc 1201 Peachtree Street 400 Colony Square Suite 200 630 Atlanta GA 30361 USA Tel 1 770 612 8014 Fax 1 678 302 7013 www dmt usa com sales dmt usa com Support dmt usa com
7. 28 So TERRE 29 5 4 In Vitro Experiment 1 Noradrenaline contractile response 30 5 5 n Vitro Experiment 2 Acetylcholine relaxation curve 30 Appendix 1 Terms of warranty 34 Appendix 2 Service check J T T T 35 Appendix Shipping instructions 36 Appendix 4 Tissue bath accessories and spare parts 37 A4 1 General Tissue bath equipmenti 37 A4 2 Tissue bath OOMO accessories 38 A4 3 Tissue bath OOMO spare parts 38 Appendix 5 Fuse replacement 39 Appendix 6 Calibration of ocular reticule 40 Appendix 7 How to read a millimetre micrometer 43 Appendix 8 Normalization theory T 43 Appendix 9 System specifications
8. 45 DMT Tissue Bath System Model 00MO About this manual This manual contains a complete list of procedures describing how to install maintain and get started using the Tissue bath system Model 7OOMO version 2 2 Chapter 1 provides an overview of the construction and basic features of the Myo In terface and the Tissue bath unit Chapter 2 describes step by step how to set up a complete OOMO Tissue bath sys tem including accessories Chapter 3 is a complete manual to the Myo Interface The chapter describes in detail the construction of the menu system and how to use all the features of the Tissue bath system Chapter 4 contains procedures describing general as well as daily maintenance of the Tissue bath unit e g adjustment of supports weight calibration of the force transducer and cleaning instructions Chapter 5 describes how to get started using the Tissue bath system This includes a discussion about normalization and a few example pharmacological experiments which are suitable for studies with large blood vessels Appendices contain additional information about normalization theory ocular calibra tion service shipping instructions system specifications equipment lists accesso ries and spare parts and fuse replacement User manual 700MO About this manual 10 Unpacking the Tissue bath system Please take a few minutes to carefully inspect your new Tissue Bath System for damage which may have occurre
9. chamber and 3 uM cocaine add 15 uL of 10 M to 5 mL PSS in chamber for at least 10 minutes 4 Add increasing concentrations of noradrenaline into the bath use Table 6 1 as a guideline Wait for a stable contractile response or a standard time such as 2 minutes between each application 5 5 In Vitro Experiment 2 Acetylcholine relaxation curve The purpose of the present protocol is to determine the sensitivity of the endothelium dependent vasodilator acetylcholine in noradrenaline pre contracted rat mesenteric small arteries Background Acetylcholine causes relaxation of rat mesenteric small arteries by activating of mus carinic M3 receptors at the endothelial cell layer leading to release of endothelium derived relaxing factors DMT Tissue Bath System Model 00MO 03 1ybof1DM 05 41 X 7 In M the NA in the myograph chamber the L volume of no radrenaline is ignored Table 5 1 Suggested concentrations of noradrenaline to add to 5 mL PSS Rat mesenteric arteries do not show spontaneous tone in the myograph which is why it is necessary to first induce a contraction to be able to observe the relaxation to ace tylcholine In this protocol the contraction is induced by noradrenaline The required concentration of noradrenaline needs to be optimised since a too low concentration makes it impossible to evaluate the relaxation On the other hand it may be difficult to relax
10. is to determine the contraction sensitivity to noradrenaline the vasodilatory effect of noradrenaline is eliminated throughout the experiment by the constant presence of the B adrenoceptor antagonist propranolol Rat mesenteric arteries are densely innervated by sympathetic nerves which have a highly efficient reuptake mechanism that removes noradrenaline from the neuromuscular junction The reuptake mechanism will create a concentration gradient between the solution around the vessel segment and the receptors on the smooth muscle To correctly determine the sensitivity to noradrenaline it is necessary to eliminate this concentration gradient by performing the experiment in the presence of cocaine to block the noradrenaline reuptake To determine the sensitivity to noradrenaline the vessel segment is exposed to increasing concentrations of noradrenaline Each concentration is applied until a steady response has been reached and then the next concentration is applied When the vessel segment is fully contracted or does not response more upon increasing the noradrenaline concentration the experiment is ended Protocol Prepare the following stock solutions e Noradrenaline 10 10 107M e Propranolol 10 M e Cocaine 10 M 1 Mount and normalise the vessels as described in Chapter 5 2 Perform a standard start as described at start of this chapter 3 Incubate the vessel segment in 1 uM propranolol add 5 uL of 10 M to 5 mL PSS in
11. maximum force value can be changed by firstly making the channel of interest active and then increasing or decreasing the value using the or buttons Irrespective of which maximum force value is entered the analog output is fixed to 1 V at max Calibration menu The calibration menu permits calibration of the transducers using the calibration kit provided with the 610M system The F1 button changes which transducer is active in the list Slct stands for select Pressing the F2 button will begin the calibration process for the force transducer selected indicated by gt Calibration of the transducers is described in full in Chapter x Setup menu The Setup menu provides access to submenus which are used to change the gain setting serial port connection and link if multiple systems are connected To enter the desired submenu use the or buttons and when the appropriate number is shown the submenu is activated by pressing F2 Whenever any changes have been made one must return to the Setup menu using T for the changes to take effect DMT Tissue bath system Model 00MO Act temp Seb temps Heat is ON Force 1 OUT Force 2 OUT Force 3 OUT Force 4 OUT Calibrate 1 Calibrate 2 Calibrate 3 Calibrate 4 Valve delay submenu The valve delay submenu is found under Setup submenu 3 By default the valve delay is set to 1 second the available delay range is 1 to 99 seconds The
12. super maximally contracted arteries which may lead to an underestimation of the sensitivity to acetylcholine Therefore it is recommended to apply a concentration of noradrenaline inducing 60 70 of maximal contraction response In practice this concentration is found by performing a noradrenaline concentration response curve as described in the previous section The vessel segment is exposed to the noradrenaline concentration and when the response has stabilised increasing concentrations of acetylcholine are added to relax the vessel Each concentration is applied until a steady response has been reached and then the next concentration is applied When the vessel segment is either fully relaxed or does not relax more upon increasing the acetylcholine concentration the experiment is ended Protocol Prepare the following stock solutions e Acetylcholine 104 10 102 M e Noradrenaline 102 M Mount and normalise the vessels as described in Chapter 5 2 Perform a standard start and check the vessel segment for endothelium func tion as described at the start of this chapter 3 Add noradrenaline to obtain a response around 60 of maximum determined from the previous noradrenaline concentration response curve When the con tractile response is stable add increasing concentrations of acetylcholine to the chamber using Table 6 2 as a guideline Wait for a stable contractile response or a standard time such as two minutes between
13. the force values in millinewtons mN Removing and replacing a chamber unit When a chamber unit is unplugged from the Myo Interface the force display does not show a force value An example display is depicted to the right where myograph unit 1 is unplugged To replace the unit firstly press the arrow displayed in the active line The display now changes to Now mount myo No 1 Put the chamber unit in place on the interface Note Always place chamber unit one at No 1 on the interface unit two on No 2 and so forth and plug the chamber cable into the corresponding input on the interface s rear panel If the unit is not plugged into the interface within 10 seconds then the arrow key should be pressed again the interface will display the previous message Once the unit is connected the active line will again display a force reading Zero menu This menu is used to zero the output of the transducers The F1 button changes which transducer is active in the list Slct stands for select Pressing the F2 button will zero the selected trans ducer indicated by gt After selecting F2 the selected transducer will be zeroed and User manual 700MO Tissue Bath System Model 700MO Version 2 2 Force 1 Force 2 Force 3 Force 4 PUSH f BEFORE MOUNT Force 2 1 03 mN Force 3 1 87 mN Force 4 0 23 mN NOW MOUNT MYO NO 1 Force 2 1 03mN Force 3 1 8 7 mN Force 4 0 23mN gt Zero 1 Zero 2
14. the gas input tube on the rear panel of the interface The needle valves are used to regulate the level of bubbling to each chamber Each needle valve has a lock device attached Note To ensure longevity the needle valves should be turned regularly fully out and in a few times and greased to prevent them from becoming stiff or fixed use the linear grease provided with the system 3 Suction connection The system has an inbuilt aeration manifold with separate valves that allow each chamber to be drained individually After connecting your suction source to the interface the appropriate chamber will be drained by pressing the associated numeric button The suction pipes are inserted into the chambers by gently pull ing the pipe up turning it 90 counter clockwise and releasing it into the chamber fig 2 2 Note When draining the chambers using the automatic suction function continu ing to press the button for an additional 3 5 seconds after the chamber appears to be empty will ensure no leftovers from the previous solution are retained in the tubing and valves e Funnel e For drug application Temperature probe Figure 2 2 Suction connection Figure 2 3 Chamber cover 4 Chamber covers The chamber covers are used to keep the temperature and other conditions gas tension pH of the solution surrounding the mounted segment as accurate as possible There are holes in the covers which serve multiple functions fig 2 3 an
15. 1 17 0 29 KH PO 136 09 1 18 0 16 Calcium free Physiological Saline Solution Ca free PSS To make 1 L of Ca free PSS Use the recipe for regular PSS but omit the CaCl and add EGTA to buffer the residual Ca in solution The concentration of EGTA in the PSS should be 1 5 mM to ensure sufficient buffering User manual 700MO Chapter 5 33 34 Appendix 1 Terms of warranty Warranty DMT A S warrants to the original user that myograph systems manufactured by DMT A S will be free from defects in materials and workmanship for a period of three years after the date of delivery DMT A S will repair or replace any defective part subject to the conditions limitations and exclusions Exclusions Force and pressure transducers separately or part of myograph systems manufac tured by DMT A S are disclaimed from any warranty Limitations This warranty shall not apply to equipment subjected to accidental damage improper use alteration or deterioration Warranty on third party products will be as determined by their respective manufac turer DMT A S shall not be liable for consequential incidental special or other direct or indirect damages resulting from economic loss or property damage sustained by you or any end user from the use of the products sold or services rendered hereunder Warranty Returns A Return Material Authorisation RMA number is required for all returns This number should be clearly indicated on all re
16. 2003 measurement control and laboratory use EN 61010 1 Corr 1 2003 Part 1 General requirements EN 61010 2 101 2003 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 101 Particular requirements for in vitro diagnostic IVD medical equipment EN 61326 2 6 2005 Electrical equipment for measurement control and laboratory use EMC requirements Part 2 6 Particular requirements In vitro diagnostic IVD medical equipment With reference to regulations in the following direc tives 2006 95 EC 89 336 EEC User manual 700MO Certificate of Conformity Contents IGICUR T E LT 3 DEVE OG GUN ON 4 S 2 5 6 PAE OV u u u uuu uu 6 Certificate of Conformitty J anam aan 7 About this manual J SE RENI RAE 9 Unpacking the Tissue bath system 10 Chapter 1 System overview 11 JL WIV ORES aCe front pane LIU A A l l lll i ii 11 1 2 Myo Interface rear panel a a
17. 4 1 Change and adjustment of mounting supports The chamber units can accommodate mounting supports of various sizes As the mounting supports are readily changed it is easy to perform experiments with differ ent vessel sizes The mounted supports will require adjustment with time due to continuous use of the myograph system and repeated greasing of the transducer pinhole Note The transducers are fragile and sensitive to mechanical strain Be very cautious not to put strain on the transducer when changing or adjusting the mounting sup ports Changing supports fig 4 1 1 Loosen screw A to align support vertically 2 Loosen screw B C to move support back or forward 3 Loosen the screws on the side of the supports to adjust as necessary horizontal Figure 4 1 Changing supports Changing the pins fig 4 2 4 Rewind the micrometer positioner all the way back 5 Loosen the screw A close to the transducer and carefully pull the head with the female part 6 Loosen the screw B on the micrometer side and perform the same procedure li A Figure 4 2 Changing pins User manual 700MO Chapter 4 19 20 4 2 Force transducer calibration As a part of the general maintenance of the myograph DMT recommends that the myograph is weight calibrated at least once every month DMT also recommends that the myograph is weight calibrated every time the system has been moved or has not been used for a long period
18. O solution for about 15 minutes 5 Wash the myograph chamber and supports several times with double distilled water Important Notes Be very careful using step and 4 repeatedly as strong reagents may damage the myograph unit e After cleaning ALWAYS check that the greasing around the transducer pin is suf ficient to keep the buffer solution away from the transducer compartment In cases of red or brown discolorations appearing on the chamber sides or on the supports the following cleaning procedure will work in most cases 1 Incubate the myograph chamber and supports for 30 minutes with 20 ul of a 2 mM T 1210 Tetrakis 2 pyridylmethyl ethylenediamine solution dissolved in double distilled water 2 Use a cotton swab stick to mechanically clean all the affected surfaces during the last 15 minutes of the incubation period 3 Wash the myograph chamber and supports several times with double distilled water 4 Incubate the myograph chamber with 96 ethanol for 10 minutes while continu User manual 700MO Chapter 4 25 26 ing the mechanical cleaning with a swab stick 5 Remove the ethanol solution and wash a few times with double distilled water Incubate the myograph chamber and supports with an 8 acetic acid solution for 10 minutes and continue the mechanical cleaning with a swab stick 6 Wash the myograph chamber and supports several times with double distilled water Important n exceptional cases it may
19. Tissue Bath System Model 7OOMO User Manual Version 2 2 RAN Sse T ECHANOL LOGS r l e 5r P4 DMT Tissue bath system Model 00MO Tissue Bath System Model 0DOMO User manual Trademarks PowerLab and LabChart are registered trademarks of ADInstruments Pty Ltd The names of specific recording units such as PowerLab 4 25 are trademarks of ADInstruments Pty Ltd Pentium is a registered trademark of the Intel Corporation Windows Windows 95 Windows 98 Windows ME Windows NT Windows 2000 and Windows XP are registered trademarks of Microsoft Corporation All other trademarks are the properties of their respective owner DMT reserves the right to alter specifications as required This document was as far as possible accurate at the time of printing Changes may have been made to the software and hardware it describes since then New information may be supplied separately This documentation is provided with the DMT Tissue bath system Model 7OOMO Version 2 2 Document Number 7OOMO UG2 2A No part of this document may be reproduced by any means without the prior written permission of DMT Copyright 2008 DMT A S M DANISH MYO TECHNOLOGY DMT A S DMT Asia DMT USA Inc Skejbyparken 152 Everwin Gardens 1201 Peachtree Street DK 8200 Aarhus N Rm 502 Block B 400 Colony Square Suite 200 630 Denmark 521 Wanping Nan Lu Atlanta GA 30361 Shanghai 200030 USA Chi
20. about 10 seconds continuously press the valve button down Turn off the vacuum pump and then the oxygen supply Remove any buffer remaining on the outside of the pipes with absorbant paper Force transducer The force transducer is the most delicate and fragile component of the myograph system and it should therefore be handled with the utmost care One of the jaws in each myograph is connected to the transducer pin The transducer pin is attached to the myograph mounting supports and is located outside of the chamber as illustrated in fig 4 7 Despite there being no direct contact to the salt solution in the chamber there is a risk for evaporating solution to deposit calcium in the pinhole The hole running into the transducer house the hole is therefore filled with high vacuum grease Figure 4 7 Transducer pin hole to be sealed up with high vacuum grease red arrows indicate points where grease should be checked and replaced As a part of daily maintenance it is very important to inspect the greasing of the trans ducer hole before starting any experiment Insufficient greasing permits salts and fluid to enter thereby causing damage and malfunction of the force transducer Important DMT recommends with frequent use of the myograph that the high vacuum grease sealing up the transducer hole is checked and if necessary replaced once a week e DMT takes no responsibilities for the use of any other kinds of high vacuum grease tha
21. als are required The converter if external must be placed between the computer and the first myograph Note For further information please contact DMT Linking multiple systems Connect a serial cable to the computer and to the signal converter Connect a second serial cable to the other end of the signal converter and the other end of the cable to the RS485 plug on the myograph system that will be 1 Connect a serial cable to the other RS485 plug on the interface Other end of cable must be connected to system 2 Repeat this if 3 or 4 systems are linked together Important When using the serial connection the systems must always be linked as follows system 1 to 2 2 to 3 and 3 to 4 Valve delay 1 sec Force Range 200mN Serial port RS232 gt Serial port RS485 gt Serial port RS485 Myograph no User manual 700MO al Chapter 3 17 18 When all systems are linked together in the above order enter submenu 10 Press to change serial port to RS485 Use F1 to move the active line marker gt down and select the system number equivalent to the way in which the serial cables have been connected Exit the serial port menu by pressing f DMT Tissue bath system Model 00MO Chapter 4 The Tissue bath unit This chapter contains a complete explanation of how to adjust calibrate and main tain the Tissue bath 7OOMO units to ensure the equipment performs to the highest standard
22. ation and prices A4 1 General Tissue bath equipment This section contains a complete checklist of laboratory equipment needed when set ting up a Tissue Bath system Dissection stereo microscope Including ocular micrometer and stage microm eter DMT recommends the Zeiss Stemi 2000 Stereo Microscope Mounting forceps DMT Item DF 3000 DMT recommends Dumont Medical No 5 tip 0 10 mm x 0 06 mm Dissection scissors DMT Item 05 1000 DMT recommends Geuder G 19745 8 cm straight trabeculum Pipettes DMT recommends u pipettes Light source DMT recommends Schott Cold Light Source either Model KL 200 or Model KL 1500 LCD Water bath including heater DMT recommends Julabo 5 L open bath circulator with plexiglass bath tank Glass bottle 2 L DMT recommends a 2 L thick walled glass bottle to collect the used solution from the myographs and act as a suction trap to prevent fluid entering the vacuum pump Vacuum pump DMT recommends a membrane vacuum pump having a volume of at least 9 L minute Dissection petri dish DMT Item PD 2000 DMT recommends a 9 cm glass Petri dish coated with a 5 mm Sylgard polymer layer User manual 700MO Appendix 4 31 38 A4 2 Tissue bath 700MO accessories This section contains a list of special accessories available for the Tissue Bath system ADInstruments PowerLab data acquisition system Including LabChart data acquisition and a
23. be necessary to demount the supports and clean them and the myograph chamber seperately to ensure that all surfaces are cleaned DMT Tissue bath system Model 00MO Chapter 5 Getting started 5 1 Normalization The importance of normalising the preparation is three fold 1 Experiments with elastic preparations like vessels can only have meaning if they are performed under conditions where the size is clearly defined 2 Clearly defined conditions are required in pharmacological experiments as the sensitivity of preparations to agonists and antagonists is dependent on the amount of stretch 3 The active response of a preparation is dependent on the extent of stretch which makes it important to set the preparation to an internal circumference giving maximal response The aim of the normalization procedure is to stretch the segment to a so called normalized internal circumference IC defined as a set fraction of the internal cir cumference IC that a fully relaxed segment would have at a specified transmural pressure For small rat arteries the target transmural pressure is typically 100 mmHg 13 3 kPa For other ring preparations such as vas deferens or ileum this procedure may not be necessary In which case stretching the vessel to a pre defined level of force may be sufficient Principles of the normalization procedure In practice the normalization is performed by distending the segment stepwise and measuring sets o
24. cular scale 4 Calculate the conversion factor 5 Mean Stage Micrometer Length mm Mean Reticule Scale div User manual 700MO Appendix 6 41 42 Appendix How to read a millimetre micrometer Sleeve scale Thimble scale Figure A7 1 Overview of the micrometer parts actual reading 20000 um 20 mm Sleeve scale The micrometer sleeve scale has a total length of 25 mm divided into fifty equal parts Each part of division above the horizontal line represents 1 mm where each fifth line is marked by a longer line and a number which designates the length in mm Each part of division below the horizontal line is placed between each 1 mm mark scale above the horizontal line and represents 0 5 mm Thimble scale The thimble is divided into fifty equal parts and one complete rotation of the thimble Is indicated by the smallest division on the sleeve which equals 0 5 mm Each divi son on the thimble scale is 10 um If the thimble scale falls between two lines then a number between O and 10 um must be approximated Example 1 1 Note that the thimble has stopped at a point be yond 10 on the sleeve indicating 10000 um 10 mm 2 Note that there is no mark completely visible be tween the 10 mm mark and the thimble Read the value on the thimble corresponding to the intersection with the horizontal line on the ia mx Figure A7 2 Example 1 reading 10380 um sleeve A Reading on sle
25. d during handling and shipping If you suspect any kind of damage please contact DMT immediately and the matter will be pursued soon as possible If the packing material appears damaged please retain it until a possible claim has been settled We recommend that you store the packing material for any possible future transport of the Tissue bath system In case of transport and the original packing material is unavailable please contact DMT Sales Department for advice and packing instructions After unpacking your new Tissue bath system please use the following list to check that the system is complete 1 interface unit e 4chamber units with mounted pin supports 200 um 4 chamber covers 1external temperature probe 1 power cord e 1 calibration kit including bridge balance and 2 gram weight 4 plastic funnels 1tube of high vacuum grease 1 tube of grease for linear slides e 2 Allen keys 1 small screwdriver e 1 DMT Tissue bath OOMO system User manual 1 manual by Professor M J Mulvany Procedures for Investigation of small vessels using small vessel myograph The shape of the AC plug varies by country be sure that the plug has the right shape for your location DMT Tissue bath system Model 00MO Chapter 1 System overview 1 1 Myo Interface front panel VALVE VALVE VALVE T e Valve ke S Power indicator y Myo Interface display
26. d slots for the mounting supports and suction gas tubes 2 3 The first weight calibration Prior to the shipment of the Tissue bath 7OOMO system it has gone through two days of continuous testing including a final weight calibration However in order to ensure that the myograph is working at highest performance DMT recommends that a new weight calibration is performed before starting to use the myograph system The weight calibration procedure is described in detail in Chapter 4 DMT Tissue bath system Model 00MO Chapter 3 The Myo Interface 3 1 Turning on the Myo Interface When the Tissue Bath 7OOMO is switched on the start up message depicted to the right is shown After a few seconds during which the system autocalibrates the A D converters pressing one of the arrow buttons will display the force menu 3 2 Main menus and submenus General navigation The various menus are selected the data changed and values entered with the F1 F2 and the arrow buttons The F1 and F2 button functions differ according to the current menu while the arrow buttons typically have the following functions lt Increase and decrease the data in the active line respectively f Scroll up and down in the menus At the top line of a menu use f to change to the previous main menu The active line in the menu is indicated by a symbol displayed in the left side of the display Force menu The force menu provides an online reading of
27. e voltage selector block Note ensure that the correct voltage for your country is displayed EP sss IE Voltage selector block User manual 700MO Appendix 5 39 Appendix 6 Calibration of ocular reticule Principles of ocular calibration The purpose of calibrating the eyepiece reticule is to determine a conversion factor 6 allowing the microscope to be used for measuring ring segment lengths mounted in the tissue bath system Several types of eyepiece reticules are available for such a purpose The most simple and yet very useful type is a horizontal scale as illustrated in fig A6 1 e 70 30 91 Xm 3 a4 50 m Figure A6 1 Horizontal eyepiece reticule scale The basic principle is to use the eyepiece reticule typically consisting of 50 100 divi sions to measure the length of an object in terms of reticular divisions spanned by the object Having the conversion factor specific for the eyepiece reticule and magnifi cation used the length of the object in millimetres is easily calculated All reticules need to be calibrated in order to determine the conversion factor char acteristic for that specific eyepiece reticule and the magnification used For such purpose a stage micrometer is needed A stage micrometer is simply a microscope glass slide with a scale engraved on the surface A typical micrometer scale is 2 00 mm long engraved with divisions of 0 01 mm equalling 10 per division Howev
28. each application User manual 700MO Chapter 5 31 03 J iuofiDM 05 of 08M 7b5yLofiM 5 Auf M In calculating the ACh in the myograph chamber the applied volume of ACh is ignored Table 5 1 Suggested concentrations of acetylcholine to add to 5 mL PSS 5 6 Buffer Recipes Physiological Saline Solution PSS To make 1 L of PSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 118 99 6 95 KCI 14 56 4 69 0 35 MgSO 7H 0 246 48 1 17 0 29 KH PO 136 09 1 18 0 16 Solution 2 Chemical MW g mol Conc mmol L Conc g L CaCl 2H O 147 02 2 50 0 37 Solution 3 Chemical MW g mol Conc mmol L Conc g L NaHCO 84 01 25 00 2 10 EDTA 372 24 0 03 0 01 Glucose 198 77 5 50 1 09 1 Dissolve the chemicals in approximately 100 mL double distilled H O as three individual solutions as described in the table above Gently heat solution 3 to dissolve the EDTA Solution 1 is added to a graduated bottle and the bottle is filled with double distilled H O to a final volume of 500 mL Solution 3 is added to the graduated bottle which afterwards is filled with additional double distilled H O to a final volume of about 850 mL Aerate the solution with carbogen 95 O 5 for about 20 minutes Solution 2 is added and the graduated bottle is filled with additional double distilled H O to reach the final volume of 1000 mL Continue the carbogen bubbling until the pH of the buffe
29. ems in the middle of the box and fill out the surroundings with chips of expanded polystyrene Important Ensure before closing the box that none of the enclosed items are loose as transport by road or air from time to time can be quite rough Address the box to DMT A S Skejbyparken 152 DK 8200 Aarhus N Denmark Make sure that all four sides of the box are marked fragile or similar Make an indi cation on the top of the box that it contains goods returned for repair service Customers outside the EC must further enclose a pro forma invoice stating that the box contains goods being returned for repair or service If arranging transportation through a courier please keep in mind the high value of the myograph system and that a standard insurance provided by the courier in most cases is insufficient to cover damage or loss of the myograph system In most cases an additional insurance coverage is needed DMT Tissue bath system Model 00MO Appendix 4 Tissue bath accessories and spare parts This appendix contains a complete register of equipment needed to set up a Tissue bath system In addition a list of special Tissue bath OOMO accessories and spare parts is included here Besides the main focus on development and manufacturing DMT has specialised in offering our costumers first class laboratory equipment needed for a Tissue bath setup at very competitive prices Please contact DMT Sales Department for further product inform
30. er micrometer glass slides with less fine divisions are also useful for calibrating the ster eomicroscope to be used with the myograph Ocular calibration procedure 1 Decide which microscope magnification is to be used for the segment length measurements Use the largest possible fixed magnification where the eyepiece reticule scale still covers the whole gap of the myograph jaws 2 Place the stage micrometer on the microscope stage and focus on it Fit one of the division lines on the stage micrometer to one of the division lines of the reticule scale very precisely While keeping the stage micrometer absolutely fixed on the microscope stage find another position on both scales where the division lines also fit precisely Read the position of the two fit points on both scales and fill in the values in the Ocular Calibration Sheet Repeat the procedure twice DMT Tissue bath system Model 00MO Ocular calibration sheet Stage micrometer Reticular scale Calculations 1 Calculate the length between the two positions on the stage micrometer by sub tracting the value of position 1 from the value of position 2 Multiply the length in divisions with the length of each division to get the length in mm 2 Calculate the length between the two positions on the reticule scale by subtracting the value of position 1 from the value of position 2 3 Calculate the mean length value of both the stage micrometer and the reti
31. eve 10000 um B No additional mark visible O um C Thimble reading 380 um Total reading 10380 um Example 2 1 Note that the thimble has stopped at a point beyond 16 on the sleeve indicating 16000 um 16 mm 2 Note that this time a mark is visible between the 16 mm mark and the thimble indication 500 um 3 Read the value on the thimble corresponding to the intersection with the horizontal line on the sleeve Figure A7 3 Example 2 reading 16780 um A Reading on sleeve 16000 um B One additional mark visible 500 um C Thimble reading 280 um Total reading 16780 um DMT Tissue bath system Model 00MO Appendix 8 Normalization theory The importance of making a normalization before initiating an experiment with any tubular tissue segment is described in Chapter 5 In this appendix the mathematical rationale and calculations underlying the normalization procedure are described in detail Mathematical calculations Let X Y be the pair of values representing the micrometer reading and force read ing respectively characterising each step in the normalization procedure Y is the force reading at the start position of the normalization procedure where the wires are just separated and the force reading is approximately zero Then given that tension on the vessel is equal to force divided by wall length the wall tension at the i th mi crometer reading is calculated by o 25 a a where is the micro
32. f micrometer and force readings fig 5 1 step 1 4 These data are converted into values of internal circumference um and wall tension T mN mm re spectively Plotting wall tension against internal circumference reveals an exponential curve and by applying the isobar curve corresponding to 100 mmHg IC is calculat ed from the point of intersection using the Laplace relation fig 5 2 IC is calculated from IC by multiplying a factor giving an internal circumference at which the active force production as well as the sensitivity to agonists of the segment is maximal For rat mesenteric arteries the factor is O 9 but both this factor as well as the transmural pressure has to be optimised for each particular segment The normalized internal diameter is calculated by dividing IC with m Appendix 8 contains a complete description of the mathematical rationale and cal culations of the normalization procedure gt N p Force mN mm 100 200 500 600 700 900 Internal Circumference pm Time min Figure 5 2 lllustration of the expo nential curve fittin and determina tion of IC oo Figure 5 1 Illustration of the stepwise normalization procedure User manual 700MO Chapter 5 21 5 2 Standard start The purpose of performing a standard start is to 1 Re activate the mechanical and functional properties of the vessel segment 2 Check that responses to different types of stimuli are normal
33. global customer base our number one goal is to develop and manufacture first class research equipment within the fields of physiology and pharmacology DMT Tissue bath system Model 00MO Safety The Tissue bath system has been designed for use only in teaching and research applications It is not intended for clinical or critical life care use and should never be used for these purposes nor for the prevention diagnosis curing treatment or alleviation of disease injury or handicap Do not open the unit the internal electronics pose a risk of electric shock Do not use this apparatus near water e To reduce the risk of fire or electric shock do not expose this apparatus to rain or moisture Objects filled with liquids should not be placed on the apparatus Do not block any ventilation openings Install in accordance with the manufacturer s instructions Do not install near any heat sources such as radiators heat registers stoves or other apparatus that produce heat e Only use attachments and accessories specified by the manufacturer e Unplug this apparatus during lightning storms or when unused for long periods of time e This apparatus must be grounded e Use a three wire grounding type cord similar to the one supplied with the product e Do not defeat the safety purpose of the polarized or grounding type plug A polarized plug has two flat blades one being wider than the other A grounding type plug ha
34. h out KPSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 3 Wash out PSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 4 Wash out KPSS 4 x with PSS Stimulate for 3 minutes Wait 5 minutes Stimulus 5 Wash out KPSS 10 uM NA 4 x with PSS Stimulate for 3 minutes Ready for experiment DMT Tissue bath system Model 00MO 5 3 Endothelium function The reasons for checking endothelium function may include 1 To check whether the relaxing function of the endothelium is intact The procedure is performed to make sure that the endothelium is not damaged during the dissec tion or mounting procedure 2 If an experiment requires removal of the endothelium this procedure is useful to check whether the endothelial cells were successfully removed The procedure can be performed after the vessel segment has been heated equili brated and normalized Preferably the procedure should be done after performing a standard start to make sure that the vessel segment is viable The present procedure is for use with rat mesenteric arteries Another procedure may be needed for other animal species and tissue or vessel types Principles of checking endothelium function Stimulating a vessel segment with acetylcholine causes a release of nitric oxide NO also known as EDRF from the endothelium cells and subsequent relaxation of the vascular smoth muscle cells
35. in appearance and thereby ensuring that the functionality of the vessel segment has not been dam aged during the dissection or mounting procedures 3 Ensure that the tension development gives an effective active pressure that is above the chosen accepted value usually 13 3 kPa 100 mmHg The standard start is performed after the vessel segment has been heated equili brated and normalized The present procedure is suitable for rat mesenteric arter ies Another procedure may be needed for other animal species and tissue or vessel types Principles of the standard start procedure The standard start procedure consists of a series of five stimuli and washout periods The first two stimuli are performed using a mixture of KPSS and 10 uM noradrena line to give a maximum contractile response The third stimulus is performed using a mixture of PSS and 10 uM noradrenaline to give a maximum pure agonist mediated o adrenoceptor contraction The fourth stimulus is performed using KPSS to give a depolarising contractile response this stimulus also includes a component from neurally released noradrenaline The final stimulus is performed using a mixture of PSS and 10 uM noradrenaline All solutions are preheated to 37 C and aerated with a mixture of 95 O and 5 CO before use Instructions for making the necessary solutions are described at the end of this chapter Procedure for a standard start Repeat 1 x Stimulus 1 amp 2 Was
36. n the one available from DMT e DMT takes no responsibilities for any kind of damage applied to the force trans ducer Linear slides Check the linear slides under the black covers for grease at least once a week In case of insufficient lubrication grease the slides with the Grease for Linear Slides enclosed with the myograph Apply the linear slide grease at the places marked by the arrows in fig 4 8 DMT Tissue bath system Model 00MO Figure 4 8 Greasing points on the linear slides Myograph cleaning DMT strongly recommends that the myograph chamber and surroundings are cleaned after each experiment At the end of the experiment use the following procedure to clean the myograph chamber and supports 1 Fill the myograph chamber to the edge with an 8 acetic acid solution and allow it to work for a few minutes to dissolve calcium deposits and other salt build up Use a swab stick to mechanically clean all chamber surfaces 2 Remove the acetic acid and wash the myograph chamber and supports several times with double distilled water 3 If any kind of hydrophobic reagent have been used which might be difficult to remove using step 1 and 2 then try incubating the chamber and supports with 96 ethanol or a weak detergent solution 4 To remove more resistant or toxic chemicals incubate the myograph chamber and supports with 1 M HCI for up to 1 hour In exceptional cases incubate the chamber and supports with an up to 3 M HN
37. na Tel 45 87411100 86 0 21 64869685 Tel 1 770 612 8014 Fax 45 87 41 11 01 Fax 86 0 21 64280591 Fax 1 678 302 7013 www dmt dk www dmt asia com www dmt usa com sales dmt dk sales dmt asia com sales dmt usa com support amp dmt dk Support dmt asia com Support dmt usa com User manual 700MO Trademarks Introduction Until the mid 1970s most of the details about the mechanical morphological and pharmacological properties of vascular smooth muscle was obtained from studies on relatively large vessels At that time rat tail arteries were the smallest vessels to be investigated in detail due to limitations in the available in vitro techniques For example studies measuring the contraction force were routinely performed with only one of the mounting wires secured Furthermore relatively large wires 100 200 um were used which precluded the use of small vessels and the vessel segment had to be directly manipulated with the dissecting equipment causing inevitable mechanical trauma Investigations of smaller vessels were therefore limited to in vivo perfusion experiments and histological examinations In 1976 Professors Mulvany and Halpern described for the first time a new technique that made it possible to investigate highly isometric responses from vessels with internal diameters as small as 100 um The mounting procedure was refined twofold both ends of each mounting wire were secured under tension without any direct mani
38. nalysis software pH Meter Including pH electrode and pH meter Standard PC system DMT Item 80150 CS 200 4 Channel Current stimulators DMT Item CS 200 Combined pulse and train generator A4 3 Tissue bath 700MO spare parts This section contains a complete list of standard spare parts available for the Tis sue Bath 7OOMO For parts not listed in this appendix or for special parts which may need to be custom made please contact DMT for further information Force transducer DMT 61780 High vacuum grease and grease for linear slides DMT Item HVG 1000 Mounting support pins 0 2 0 25 0 3 0 35 and 0 4mm DMT ltem MO 200 Calibration kit DMT Item CK 6127800 Chamber cover DMT Item 61700 40 mm funnels DMT Item F 2000 DMT Tissue bath system Model 00MO Appendix 5 Fuse replacement The main fuse of the myograph system is placed inside the power inlet on the Myo Interface When a fuse blows and needs to be changed it is imperative that the replacement fuse is equal to the one blown The 7OOMO system uses T1 6A 250 V 6 3 x 32 mm DMT recommends that both fuses in the fuse block are changed at the same time as it can be difficult to determine which fuse is blown To replace the fuses Use a small screwdriver to open the voltage selector block Remove the red fuse block Remove the existing fuses Insert the new fuses Replace the fuse block back into th
39. of the transducer arm is 4 cm the weight is 2 g both angles are 90 and the acceleration of gravity is 9 81 ms the force acting on the force transducer is F 2cm 2 g 9 81 ms sin90 4 cm sin90 r 951 s As 1 N is equal 1 kg s gt F is equal to F 9 81 mN Weight calibration procedure 1 Move the pins apart in the myograph chambers Mount a wire on the transducer side of the myograph jaws in all chambers Place the units on the interface plug in the cables and fill the chambers with double distilled water This is not required if the mounting support pins are used instead of the jaws 2 Turn on the heating in the Heat main menu of the Myo Interface The system will typically reach the target set temperature by default 37 C after 20 minutes 3 Place the calibration bridge balance and weight on myograph unit 1 as illus trated in fig 4 6 It is important that the calibration kit is pre warmed together with the myograph unit Make sure that the tip of the transducer arm on the balance is positioned protruding behind the pin illustrated in Figure 4 4 Carefully move the calibration bridge until the tip of the transducer arm is placed freely which means it does not touch the pin Figure 4 4 Weight calibration setup User manual 700MO Chapter 4 21 22 4 Once the system has warmed up to the set temperature check by placing the temperature probe into the chamber go to the Calibration menu
40. of time Principles of weight calibration Weight calibrating the force transducer is based on simple physics the net torque acting on a balance when applying a certain amount of weight The magnitude of the torque x about a point of rotation P is defined by uh t r F sing where r is the distance from the point of rotation to the point on the object where the force F is acting with the angle of 0 Applying the physics to the weight calibration setup is illustrated in fig 4 3 Figure 4 3 Thereotical principle of the weight calibration Applying the weight on the pan arm creates a net torque acting at the center of grav ity resulting in a force F acting on the force transducer The following two equations describe the forces working in the weight calibration system d uq p SI rds Wa BD Sne Sin where r is the length of the pan arm is the force acting on the pan arm when applying the weight F is equal acceleration of gravity times the mass of the weight is the length of the transducer arm and F is the force acting on the force trans ducer DMT Tissue bath system Model 00MO The net torque acting at center of gravity is constant for the weight calibration setup which makes equation 1 and 2 equal making it possible to calculate the force acting on the force transducer 79 I f asc ene m as Sind 9110 As the length of the pan arm is 2 cm the length
41. on the Myo Interface In the Calibration menu ensure Calibrate 1 is active the symbol is displayed to the left use the F1 button to scroll through the list and press F2 to initiate calibration of force transducer 1 5 Ensure that the tip of the transducer arm is not touching the mounting support pin It is imperative that the force transducer is not subjected to any force at this stage When the relative force reading in the display is stable press F2 to pro ceed with the calibration 6 Carefully place the 2 g weight on the pan as illustrated in fig 4 5 The force ap plied on the force transducer as the tip of the arm pushes against the mounted wire or support pin should mimic the stretch created by the contraction of a mounted ring preparation Wait until the relative force reading is stable then press F2 to finish the calibration Figure 4 5 Illustration of how to fit the balance between the wire and the in the support Force transducer 1 is now weight calibrated to an output of 9 81 mN The display will automatically return to the Force menu Note If the force reading is unstable or gt 0 1 mN different from 9 81 mN repeat the weight calibration If the instability continues refer to section 4 3 6 Carefully remove the weight balance and calibration bridge from myograph unit 1 and move to myograph unit 2 Repeat steps 4 6 for force transducer 2 3 and 4 4 3 Checking force transducer The myograph f
42. orce transducer is a strain gauge connected in a Wheatstone bridge The force transducer for each myograph unit is located in a separate compartment transducer house see Chapter 1 3 While this provides some mechanical protection the force transducers are still very vulnerable to applied forces exceeding 1 newton 100 gram or fluid running into the transducer compartment due to insufficient greasing of the transducer pinhole If the force reading continues to be unstable in spite of a recent weight calibration then repeat the weight calibration and note down the relative force reading values shown in the Calibration menu on the Myo Interface DMT Tissue bath system Model 00MO If the value is O or above 6500 then the force transducer is broken and needs to be changed If the value is between 1 499 or 3001 6250 then contact DMT for further instructions Important If at any time the message OFF is displayed in the Force menu on the Myo Inter face this indicates that the force transducer is broken and must be replaced In this instance or in case of other problems related to the force transducer please contact DMT for further instruction and advice 4 4 Force transducer replacement If the force transducer is broken and needs to be changed please follow this step by step replacement procedure carefully 1 2 Disconnect the myograph unit from the Myo Interface grey cable Turn the myograph unit upside d
43. own and remove the bottom plate by loosen ing the two screws A B as illustrated in fig 4 6 Carefully disconnect the force transducer plug and remove the old transducer Note how the plug is connected to the old force transducer to prevent incorrect connection of the new force transducer Figure 4 6 The two screws holding the transducer house in place Remove any remaining grease from the transducer pin left inside of the trans ducer compartment of the myograph unit Also clean the hole leading from the transducer compartment to the myograph chamber Replace the bottom plate and tighten the two Allen screws Place some high vacuum grease supplied with the system around the transduc er pin in the myograph chamber Make sure that the hole is completely sealed so that absolutely no buffer solution is able to enter the transducer compartment and damage the force transducer Important The new force transducer must be weight calibrated prior to running an experiment User manual 700MO Chapter 4 23 24 4 5 Myograph maintenance The Tissue Bath OOMO is a delicate and sophisticated piece of research equipment DMT recommend that the following sections are read carefully and that the instruc tions are followed at all times Myograph chamber pipes To prevent the pipes from being blocked by buffer salt deposits after an experiment remove the chamber cover from the myograph and turn on the vacuum pump and vacuum valve for
44. pulation of the vessel segment Segments of small vessels could now be atraumatically mounted as ring preparations in a myograph for recording of highly isometric force measurements During the late 19705 some improvements were made to the myograph and in 1981 a new dual myograph that allowed simultaneous testing of two vessels was introduced In parallel the technique became widely acknowledged resulting in a growing interest in the myograph systems In 1986 the growing demand resulted in the foundation of the private company J P Trading with the purpose of making the myograph systems commercially available worldwide At the same time J P Trading initiated a comprehensive improvement programme for the existing myograph systems as well as a development programme of new myograph systems in close co operation with Professor M J Mulvany and the University of Aarhus During the late 1980s and through the 1990s several improvements were applied to the myograph systems such as a new mechanical design a more robust transducer and new electronic systems In addition new systems were introduced like the Automatic Dual Myograph 510A the Dual Myograph 410A the Multi Myograph 610M and the Confocal Myograph 120CW In 2000 J P Trading changed its company structure and became known as DMT Today DMT is one of the world s leading designers and manufacturers of wire myographs pressure myographs culture myographs and organ tissue baths Driven by our
45. r solution reaches 7 4 DMT Tissue bath system Model 00MO 25x Concentrated PSS To make 1 L concentrated PSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 118 99 173 85 KCI 14 56 4 69 8 75 CaCl 2H O 147 02 2 50 9 20 Solution 2 Chemical MW g mol Conc mmol L Conc g L MgSO H O 246 48 1 17 1 23 KH2PO 136 09 1 18 4 02 Solution 3 Chemical MW g mol Conc mmol L Conc g L EDTA 3 2 24 0 03 0 25 1 Dissolve the chemicals for solution 1 in about 800 mL double distilled H O in a 1000 mL graduated bottle Dissolve the chemicals for solutions 2 and 3 in 75 mL double distilled in individually cylinders Gently heat solution to dissolve the EDTA 2 Solution 2 and 3 is added to solution 1 and the graduated bottle is filled with additional double distilled to reach a final volume of 1000 mL Before use Dilute the 25 x PSS stock solution 1 25 with double distilled H O 4 Add 1 091 g L Glucose 2 100 g L NaHCO Aerate the solution with carbogen 95 O 5 for at least 20 minutes If necessary wait further for the pH of the buffer to reach pH 7 4 High Potassium Physiological Saline Solution KPSS To make 1 L of KPSS Use the recipe for regular PSS but replace the desired concentration of NaCl with KCI For example to make 60 mM KPSS Solution 1 Chemical MW g mol Conc mmol L Conc g L NaCl 58 44 64 86 3 19 KCI 14 56 58 82 4 39 MgSO H O 246 48
46. s and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that interference will not occur in a particular installation If this equipment does cause harmful interference to radio or television reception which can be determined by monitoring the interference while turning the equipment off and on the user is encouraged to correct the interference by one or more of the following measures e Reorient or relocate the receiving antenna e Increase the separation between the equipment and receiver e Connect the equipment into an outlet on a circuit different to that which the receiver is connected to e Consult the dealer or an experienced radio TV technician for help Approvals Complies with the EMC standards EMC 89 336 EEC EN 61326 2 6 2005 EN 61000 3 2 Certified with the safety standards Directive 2006 95 EC EN 61010 1 2001 EN 61010 1 Corr 1 2003 EN 61010 1 Corr 1 2003 EN 61010 2 101 2003 DMT Tissue bath system Model 00MO Certificate of Conformity DMT A S Skejbyparken 152 8200 Aarhus N Denmark hereby declares its responsibility that the following product Tissue Bath System Model 7OOMO Version 2 2 is covered by this certificate and marked with CE label conforms with the following standards EN 61010 1 2001 Safety requirements for electrical equipment for EN61010 1 Corr 1
47. s two blades and a third round grounding pin The wide blade or the third prong is provided for your safety If the provided plug does not fit into your outlet consult an electrician for replacement of the obsolete outlet advised that different operating voltages require the use of different types of line cord and attachment plugs Check the voltage in your area and use the correct type See the table below Line plug according to standard 110 125V UL81 and CSA C22 2 No 42 220 230 V CEE page VII SR section 107 2 D1 IEC 83 page C4 240 V BS 1363 of 1984 Specification for 13A fused plugs and Switched and unswitched socket outlets Protect the power cord from being walked on or pinched particularly at power plugs and the point where they connect to the apparatus Refer all servicing to qualified service personnel Servicing is required when the apparatus has been damaged in any way such as the power supply cord or plug is damaged liquid has spilled onto or objects have fallen into the apparatus the apparatus has been exposed to rain or moisture does not operate normally or has been dropped User manual 700MO Safety EMC EMI This equipment has been tested and found to comply with the limits for a Class B Digital device pursuant to part 15 of the FCC rules These limits are designed to provide reasonable protection against harmful interference in residential installations This equipment generates use
48. scope eyepiece reticule calibration factor in mm per division and a and a are the vessel end points when measuring the length of the mounted vessel segment The internal circumference of the mounted vessel at the i th reading is calculated by IC ICO 2 X AJ where IC is the internal circumference of the mounted vessel when the wires are just separated and is given by d where d is the wire diameter For 40 uim wires IC 205 6 um Using the Laplace relation the effective pressure P is calculated for each pair of readings The effective pressure is an estimate of the internal pressure which is nec essary to extend the vessel to the measured internal circumference T _ 5 The stepwise distension is continued until the calculated effective pressure exceeds the target transmural pressure The target value needs to be optimised for the indi vidual tissue preparation optimal active force as determined by the length tension relationship for that tissue For rat mesenteric arteries the target transmural pres sure is normally 100 mmHg 13 3 kPa 100 mmHg IC T 100 27 User manual 700MO Appendix 8 43 44 An exponential curve is fitted to the internal circumference pressure data as illus trated in fig x in Chapter x Now the isobar corresponding to 100 mmHg is used to calculate the value from the point of interception between the function of the exponential curve and the func
49. tion of the 100 mmHg isobar The normalized internal circumference IC is calculated by multiplying the internal circumference corresponding to 100 mmHg IC p by a factor k The factor is for rat mesenteric arteries O 9 Again this value should be optimised for the particular tis sue preparation being used by a length tension curve IC k 1 y The normalized internal lumen diameter is then calculated by _ de 1 TU The micrometer reading X at which the internal circumference of the normalized ves sel is set to is calculated by IC 2 DMT Tissue bath system Model 00MO Appendix 9 System specifications Tissue size Chamber Chamber material Chamber volume Chamber suction Chamber gassing Chamber cover Force range Force resolution Micrometers Weight calibration Heating Temp range Temp resolution Temp probe Output reading Analog output Digital output Voltage 500 up to 10 mm diameter ring segments Four individual chambers Acid resistant stainless steel Max 8 mL Manual or automatic time controlled user defined Individually controlled per chamber by needle valves Supplied with connections for gassing User selectable at 200 400 800 and 1600 mN 0 01 mN Manually operated Semi Automatic Built in Ambient temp 50 C 0 1 External Force mN 1 OV F S Serial interface RS232 RS485 115 1096 50 60Hz 230
50. turned myograph systems Products damaged due to improper or inadequate packaging when returned for RMA purposes are not granted warranty coverage DMT Tissue bath system Model 00MO Appendix 2 Service check For successful studies of small blood vessels or other small tubular tissue it is im perative that the myograph is performing optimally To make sure that our customers are always dealing with first class myographs DMT offers a Myograph Service Check at a very favourable price The Myograph Service Check includes a complete separation of the system for in spection of all mechanical and electronic parts The myograph is then reassembled adjusted and finally all electronic and mechanical parts are tested Please note that the service does not include or cover replacement of transducers or other parts Please contact DMT for information about prices User manual 700MO Appendix 2 35 36 Appendix 3 Shipping instructions If the myograph system needs to be sending back for service or repair please read the following shipping instructions very carefully Before you start packing the myograph system please remember that you are dealing with very delicate equipment and therefore care must be taken DMT recommends that each part of the myograph system be wrapped individually i e with bubble wrap and placed together in a large box preferable the box you once received the myo graph system in Place the wrapped it
51. valve delay is the time the valve is open once you have released the valve button on the front panel The delay time is changed using the and buttons Force range submenu The Force Range submenu is found in Setup submenu 7 By default the force range is set at 200 mN The available ranges are 200 400 800 and 1600 mN The force range is changed using the and buttons Serial port submenu For digital storage of data the interface has an integrated RS232 port and two RS485 serial ports The RS232 serial port is used when a single system is connected to a computer and the RS485 serial ports are used for multiple connections up to four systems linked together allowing 16 force channels and four temperature readings By default the system is set to use RS232 and functions for the serial settings are found in Setup submenu 10 To switch between the two outputs use the and buttons Using the enclosed serial cable with the mounted adapter the 9 pin plug is connected to the RS232 plug of the interface and the 25 pin plug connected to the serial port COM port on the computer RS232 Serial port This serial communication protocol must be used when only one system is connected to one PC RS485 Serial port This serial communication protocol must be used when multiple systems are linked together In order to use this feature an external or internal signal converter that is half duplex and toggles RTS sign
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