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Summary of Contents for DMT 620M
- Page 1 U S E R G U I D E , VO L . 2 . 0 W I R E M Y O G R A P H S Y S T E M - 6 2 0 M...
- Page 2 WIRE MYOGRAPH - 620M USER GUIDE User Guide Version 1.0...
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Page 3: Table Of Contents
CONTENTS Chapter 1 - Wire Myograph overview ....................................3 Chapter 2 - setting up the wire myograph - 620M ................................4 2.1 Changing and adjusting the mounting supports ..............................4 2.1.1 Changing the mounting supports (figure 2.1) ................................. 4 2.1.2 Coarse adjusting the jaws for small vessels (figure 2.1) ............................4 2.1.3 Fine adjusting the jaws for small vessels (figure 2.2 and figure 2.3) ......................... -
Page 4: Chapter 1 - Wire Myograph Overview
Jaw connected to Micrometer Force transducer pin Supports Figure 1.1 Wire Myograph with close-up of chamber Access or funnel Access for temperature probe Access for drug application Figure 1.2 Chamber cover CHAPTER 1 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE... -
Page 5: Chapter 2 - Setting Up The Wire Myograph - 620M
CHAPTER 2 - SETTING UP THE WIRE MYOGRAPH - 620M 2.1 Changing and adjusting the mounting supports Each chamber can accommodate mounting supports for either small vessels (>50µm) or larger segments (>500µm).The mount- ing supports can be changed easily and experiments can be performed with different vessels of varying internal diameter. -
Page 6: Fine Adjusting The Jaws For Small Vessels (Figure 2.2 And Figure 2.3)
Jaws from top view Jaws from side view Figure 2.3 - Illustrations of properly aligned jaws (depicted on the far left) and incorrectly aligned jaws (depicted in the middle and far right). CHAPTER 2 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE... -
Page 7: Fine Adjusting The Pins For Larger Vessels (Figure 2.4 And Figure 2.5)
2.2 Calibration of the force transducer As a part of the general maintenance of the Wire Myograph , DMT recommends that the Wire Myograph is force calibrated at least once a month. The Wire Myograph should also be force calibrated every time the interface has been moved. Although lab benches are all supposedly perfectly horizontal, small differences in lab bench pitch can affect the calibration of the system. -
Page 8: Chapter 3 - Experimental Set-Up
(figure 3.1 B-C). • Fill the Wire Myograph chamber with PSS (at room temperature). See appendix 1 for example of buffer recipes. Figure 3.1 A, B and C Mounting step 1 CHAPTER 3 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE... -
Page 9: Mounting Step Two
3.1.2 Mounting step two • Using forceps to hold the handle segment, transfer excised vessel from Petri dish to the Auto Dual Wire Myograph chamber. Hold the vessel as close to the proximal end as possible and try to mount the vessel onto the wire. •... -
Page 10: Mounting Step Four
Figure 3.5 A, B and C Mounting step 5 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE CHAPTER 3... -
Page 11: Mounting Step Six
3.1.6 Mounting step six • Carefully move the jaws together while ensuring that the second mounted wire lies underneath the first one secured on the left-hand jaw (figure 3.6 A). The procedure clamps the second wire to prevent it from damaging the vessel segment when securing the wire to the right-hand jaw (connected to the transducer). -
Page 12: Principles Of The Normalization Procedure
The final stimulus is performed using a mixture of PSS and 10 μM 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 in appendix 1. WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE CHAPTER 3... -
Page 13: Endothelium Function
Repeat 1 x -- Wash out -- -- Stimulus 1 & 2 -- 4 x with PSS KPSS + 10 μM NA Wait 5 minutes Stimulate for 3 minutes -- Stimulus 3 -- -- Wash out -- 4 x with PSS PSS + 10 μM NA Stimulate for 3 minutes Wait 5 minutes... -
Page 14: In Vitro Experiment 1: Noradrenaline Contractile Response
1 μL of 10 7.5 μL of 10 1 μL of 10 2.5 μL of 10 *In calculating the [NA] in the Wire Myograph chamber, the applied volume of noradrenaline is ignored. CHAPTER 3 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE... -
Page 15: In Vitro Experiment 2: Acetylcholine Relaxation Curve
3.6 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. 3.6.1 Background Acetylcholine causes relaxation of rat mesenteric small arteries by activating of muscarinic M3 receptors at the endothelial cell layer leading to release of endothelium-derived relaxing factors. -
Page 16: Chapter 4 - Cleaning And Maintenance
CHAPTER 4 - CLEANING AND MAINTENANCE 4.1 Cleaning the Wire Myograph DMT STRONGLY RECOMMENDS THAT THE WIRE MYOGRAPH AND SURROUNDING AREAS ARE CLEANED AFTER EACH EXPERIMENT. At the end of each experiment, use the following procedure to clean the Wire Myograph. -
Page 17: Maintenance Of The Force Transducer
DMT RECOMMENDS THAT THE HIGH VACUUM GREASE SEALING THE TRANSDUCER PINHOLE IS CHECKED AND SEALED AT LEAST ONCE A WEEK, ESPECIALLY IF THE WIRE MYOGRAPH IS USED FREQUENTLY. DMT TAKES NO RESPONSIBILITIES FOR THE USE OF ANY OTHER KINDS OF HIGH VACUUM GREASE OTHER THAN THE ONE AVAILABLE FROM DMT. -
Page 18: Force Transducer Replacement
IMPORTANT NOTE CALIBRATE THE NEW FORCE TRANSDUCER BEFORE PERFORMING A NEW EXPERIMENT. Figure 4.3 - The 2 screws that secure the transducer house to the chamber CHAPTER 4 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE... -
Page 19: Maintenance Of The Linear Slides
Figure 4.4 Inside the transducer housing and close-up of transducer pin. The orange arrows in the dashed frame indicates the place that the vacuum grease needs to be applied to prevent water and buffer from damaging the transducer. 4.3 Maintenance of the linear slides Check the linear slides (under the black covers) for grease at least once a week. -
Page 20: Appendix 1 - Buffer Recipes
7.25 14.5 29.0 NaHCO (84.01) 14.9 0.625 1.25 2.50 5.00 Glucose (180.16) 1.00 2.00 4.00 EDTA (380) 0.65 0.125 0.25 0.50 CaCl (110.99) 20mL 40mL 80mL 160mL (1.0 M solution) APPENDIX 1 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE... - Page 21 1. Make a 1.0M solution of CaCl (110.99) in double-distilled H O. Filter-sterilize the calcium solution through a 0.22 μm filter. The sterilized solution can be stored in the refrigerator for up to 3 months. 2. Dissolve all the chemicals except the CaCl in approximately 80% of the desired final volume of double distilled H O while being constantly stirred.
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Page 22: Appendix 2 - Normalization Theory
, by a factor k. The factor is for rat mesenteric arteries 0.9. Again, this value should be optimized for the particular tissue preparation being used by a length-tension curve. = k •IC APPENDIX 2 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE... - Page 23 The normalized internal (lumen) diameter is then calculated by: The micrometer reading X at which the internal circumference of the normalized vessel is set to is calculated by: – IC...
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Page 24: Appendix 3 - Reading A Millimetre Micrometer
A. Reading on sleeve: 16000 µm B. One additional mark visible: 500 µm C. Thimble reading: 280 µm 16780 µm Total reading: Figure A2.3 Example 2: reading = 16780 µm APPENDIX 3 WIRE MYOGRAPH SYSTEM - 620M - USER GUIDE...
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