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DMT 630MA User Manual

Automated multi myograph system
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USER GUIDE
MYOGRAPH SYSTEM - 630MA

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Summary of Contents for DMT 630MA

  • Page 1 USER GUIDE MYOGRAPH SYSTEM - 630MA...
  • Page 2 AUTOMATED MULTI WIRE MYOGRAPH SYSTEM...

  • Page 3: Table Of Contents

    3.6 In vitro experiment 1: Noradrenaline contractile response 3.7 In vitro experiment 2: Acetylcholine relaxation curve CHAPTER 4 - CLEANING AND MAINTENANCE 4.1 Cleaning the 630MA Myograph system 4.2 Maintenance of the force transducer 4.3 Maintenance of the linear slides...

  • Page 4: Chapter 1 - Wire Myograph Overview

    CHAPTER 1 - WIRE MYOGRAPH OVERVIEW Connection to Myo-Interface Micropositioner Allen screws for fine alignment of the myograph jaws Force transducer pin Myograph jaw connected to micropositioner Myograph jaw connected to force transducer Supports Figure 1.1 Wire Myograph with close-up of chamber Funnel For drug application Temperature probe...

  • Page 5: Chapter 2 - The Multi Wire Myograph Unit

    CHAPTER 2 - THE MULTI WIRE MYOGRAPH UNIT 2.1 CHANGING AND ADJUSTING THE MOUNTING SUPPORTS Each chamber can accommodate mounting The mounting supports, therefore, whether supports for either small vessels (>30µm) or they are the jaws for wires or the pins, will need larger segments (>500µm).
  • Page 6 NOTE: Number the supports with the number of the chamber they were removed from using some kind of permanent marker. Store the supports in the provided plastic case. Numbering the supports will save time when the supports are changed again, limiting the amount of adjustments needed after each change.
  • Page 7 2.1.3 FINE-ADJUSTING THE JAWS FOR SMALL VESSELS (FIGURE 2.2 AND FIGURE 2.3) 1. Tightening Screw “D” will move the micrometer 3. Tightening both screws “C” and “A” will move side jaw downward and to the left. the micrometer side jaw straight up. 2.

  • Page 8: Calibration Of The Force Transducer

    As a part of the general maintenance of the Wire perfectly horizontal, small differences in lab bench Myograph, DMT recommends that the Wire pitch can affect the calibration of the system. The Myograph is force calibrated at least once a Wire Myograph should also be calibrated if the month.

  • Page 9: Chapter 3 - Experimental Set-Up

    CHAPTER 3 - EXPERIMENTAL SET-UP This chapter contains experimental set-up for the see Procedures for investigations of small vessels Wire Myograph. For dissection of a vessel, please using a small vessel Myograph by M.J. Mulvany. 3.1 MOUNTING PROTOCOL FOR SMALL ARTERIES The procedure involves attaching the mounting exceeds about 1600 mN (160 g).
  • Page 10 3.1.2 MOUNTING STEP TWO • Using forceps to hold the handle segment, 2. Hold excised vessel about 3 mm from transfer excised vessel from Petri dish to the the cut end with one set of forceps and Wire Myograph chamber. Hold the vessel as use the other forceps to squeeze the blood close to the proximal end as possible and try remaining in lumen out through the cut...
  • Page 11 3.1.3 MOUNTING STEP THREE • Once the vessel segment is threaded onto gap to insure no point of contact (figure 3.3 the wire, catch the free end of the wire (near- est you) with the forceps and move the jaws apart.
  • Page 12 3.1.5 MOUNTING STEP FIVE • Move the jaws apart (figure 3.5 A). Take a prevent the vessel being stretched during second wire holding it about one third down the maneuverer. Be careful not to touch the from the far end using a forceps. Align the lumen of the vessel with the end of the wire wire parallel with the vessel segment such and when pushing the wire end through the...
  • Page 13 3.1.7 MOUNTING STEP SEVEN • Secure the far end of the wire under the near procedure. In case of excess of vessel on fixing screw on the right-hand jaw. Again the the far side of the jaws then move the jaws wire is passed clockwise around the screw together again and remove excessive tissue stretching the wire as the screw is tightened...
  • Page 14 • The artery is now mounted correctly and the buffer in the chamber with fresh well can moved back onto the 630MA interface gassed warm buffer. Wait 5 min and start the with the heat turned on. When the buffer...

  • Page 15: Mounting Protocol For Larger Arteries

    250µm, 300µm or 400µm pins can be replace the mounting Jaws with Pins (see chapter purchased from DMT. 2.1). With the 630MA system a box with 4 sets of 3.2.1 MOUNTING STEP ONE • First make sure that the mounting pins is placed correctly across each other in the chambers as shown below.
  • Page 16 The artery is now mounted correctly and the well gassed warm buffer. Wait 5 min and start heat of the 630MA interface can be turned ON. the Automated Normalization as described in When the buffer in the chamber have reached Chapter 3.3.5.

  • Page 17: Normalization

    3.3 NORMALIZATION The importance of normalizing the preparation is three-fold: 1. Experiments with elastic preparations like antagonists is dependent on the amount of vessels can only have meaning if they are stretch. performed under conditions where the size is clearly defined. 3.
  • Page 18 IC with 100 mmHg, IC is calculated from the point of 3.3.2 THE AUTOMATED NORMALIZATION PROCEDURE ON THE 630MA SYSTEM Make sure the wires/pins do not touch and are NORMALIZATION  SETUP close together without touching SELECT  CHAMBER  1: SELECT SELECT  CHAMBER  2:...
  • Page 19   4. Enter the values for the Norm. Time, Wire/Pin diameter. CH1  NORM.  PARAMETERS  1 DMT have the following wire and Pin products: Norm.  Time:                    60  Sec. Steel Wire : 40µm in diameter...
  • Page 20 There can be a huge difference between for your specific artery and specie please artery type and species e.g for rat mesenteric read DMT Normalization Guide. IMPORTANT: The Norm Factor has to be found for the type of vessel and species BEFORE using the automated normalization.
  • Page 21 NORM. in the given chamber. When the a1 and a2 values   have been entered the 630MA will calculate the artery Segment Length based on the a1, a2 and Eyepiece Cal values. Check if the segment length is approx. correct. The gap in the jaws are 2.00 mm (shown with red arrows in the figure to the right).
  • Page 22 CH1  NORMALIZATION Step  no.:    1                        Time:    48 13. When all parameters are programmed, CH1  NORMALIZATION Xo:      +0.00  uM              Yo:  -­12.13  mN press Start Norm.
  • Page 23 16. If the message “Not enough steps to normalize“ CH1  NORMALIZATION appears then go into the Norm. Force parameter Step  no.:    15                        Time:    0 in the Ch1 ARTERY PARAMETER menu and lower Xo:  ...

  • Page 24: Standard Start

    3.4 STANDARD START The purpose of performing a standard start is to: 1. Re-activate the mechanical and functional dissection or mounting procedures. properties of the vessel segment. 3. Ensure that the tension development gives 2. Check that responses to different types of an effective active pressure that is above the stimuli are normal in appearance and thereby chosen accepted value...

  • Page 25: Endothelium Function

    3.5 ENDOTHELIUM FUNCTION The reasons for checking endothelium function may include: 1. To check whether the relaxing function of The procedure can be performed after the vessel the endothelium is intact. The procedure is segment has been heated, equilibrated and performed to make sure that the endothelium normalized.

  • Page 26: In Vitro Experiment 1: Noradrenaline Contractile Response

    3.6 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. 3.6.1 BACKGROUND Noradrenaline (norepinephrine) causes around the vessel segment and the receptors contraction mesenteric small...
  • Page 27 3.6.2 PROTOCOL Prepare the following stock solutions: Noradrenaline: , 10 , 10 Propranolol: Cocaine: 1. Mount and normalize the vessels as described M to 5 mL PSS in chamber) for at least 10 in the former chapters. minutes. 2. Perform a standard start as described in 4.

  • Page 28: In Vitro Experiment 2: Acetylcholine Relaxation Curve

    3.7 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. 3.7.1 BACKGROUND Acetylcholine causes relaxation of rat mesenteric it is recommended to apply a concentration small arteries by activating of muscarinic M3 noradrenaline...
  • Page 29 3.7.2 PROTOCOL Prepare the following stock solutions: Acetylcholine: , 10 , 10 Noradrenaline: 1. Mount and normalize the vessels as described the previous noradrenaline concentration- in the former chapters. response curve). When contractile response stable, increasing 2. Perform a standard start and check the concentrations acetylcholine vessel segment for endothelium function, as...

  • Page 30: Chapter 4 - Cleaning And Maintenance

    CHAPTER 4 - CLEANING AND MAINTENANCE 4.1 CLEANING THE 630MA MYOGRAPH SYSTEM NOTE: • DMT strongly recommends that the Wire Myograph and surrounding areas are cleaned after each experiment. • If any acetic acid is spilled outside the steel chamber remove it immediately with a napkin and clean with distilled water to avoid damage of the aluminum base over the years.
  • Page 31 IMPORTANT: • Be very careful using HCl or HNO because these acids may cause extreme damage to the stainless steel chambers and supports. • DO NOT USE bleach to clean the chambers. Repeated use of chlorinated solutions such as bleach and HCl will cause damage to the stainless steel parts of your myograph system.

  • Page 32: Maintenance Of The Force Transducer

    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 33 In addition, then perform a new calibration as described in if the force reading(s) appear yellow in color, chapter 3.5.1 in 630MA Myograph System User cannot be reset to zero, AND the transducer Manual. Electrically noise from the surroundings...
  • Page 34 If any other problems related to the force transducer are encountered, please contact DMT for advice or further instructions. Transducer house Figure 4.2 Illustration of the transducer house 4.2.2 FORCE TRANSDUCER REPLACEMENT If the force transducer breaks and needs to be replaced, follow this step-by-step replacement procedure carefully: 1.
  • Page 35 IMPORTANT: Calibrate the new force transducer before performing a new experiment. Figure 4.3 - The 8 screws that secure the transducer house to the chamber 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.

  • Page 36: Maintenance Of The Linear Slides

    4.3 MAINTENANCE OF THE 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” included with your system. (See figure 4.5 below).

  • Page 37: Appendix 1 - Buffer Recipes

    APPENDIX 1 - BUFFER RECIPES PHYSIOLOGICAL SALINE SOLUTION (PSS) 1. Make a 1.0M solution of CaCl (110.99) in 3. Add the appropriate volume of 1.0M CaCl double-distilled Filter-sterilize for the total volume of PSS being made (for calcium solution through a 0.22 μm filter. example, 1.6mL of 1.0M CaCl for 1 liter of The sterilized solution can be stored in the...
  • Page 38 1. Make a 1.0M solution of CaCl (110.99) 4. Bring the solution up to the final volume with in double-distilled H O. Filter-sterilize the double-distilled H O. Continue to stir the calcium solution through a 0.22 μm filter. solution until the EDTA is fully dissolved. This The sterilized solution can be stored in the takes about 15 minutes at room temperature.

  • Page 39: High Potassium Physiological Saline Solution (Kpss)

    HIGH POTASSIUM PHYSIOLOGICAL SALINE SOLUTION (KPSS) 1. Make a 1.0M solution of CaCl (110.99) in 3. Add the appropriate volume of 1.0M CaCl double-distilled Filter-sterilize for the total volume of PSS being made (for calcium solution through a 0.22 μm filter. example, 1.6mL of 1.0M CaCl for 1 liter of The sterilized solution can be stored in the...

  • Page 40: Appendix 2 - Normalization Theory

    APPENDIX 2 - NORMALIZATION THEORY The importance of making a normalization before initiating an experiment with any tubular tissue segment is described in chapter 3.2. In this appendix the mathematical rationale and calculations underlying the normalization procedure are described in detail. MATHEMATICAL CALCULATIONS Let (Xi , Yi ) be the pair of values representing the micrometer reading (see appendix 3) and force reading respectively characterizing each step in the normalization procedure.
  • Page 41 The stepwise distension is continued until the calculated effective pressure exceeds the target transmural pressure. The target value needs to be optimized for the individual tissue preparation (optimal active force as determined by the length-tension relationship for that tissue). For rat mesenteric arteries the target transmural pressure is normally 100 mmHg (13.3 kPa): = 100 mmHg•...

  • Page 42: Appendix 3 - Reading A Millimetre Micrometer

    APPENDIX 3 - READING A MILLIMETRE MICROMETER Sleeve scale Thimble scale Figure A3.1 Overview of the micrometer parts (actual reading 20000 µm = 20 mm) Sleeve scale Thimble scale The thimble is divided into 50 equal parts, and The micrometer sleeve scale has a total length of 25 one complete rotation of the thimble is indicated mm divided into 50 equal parts.
  • Page 43 Example 1 1. Note that the thimble has stopped at a point beyond “10” on the sleeve indicating 10000 µm (10 mm). 2. Note that there is no mark completely visible between the 10 mm mark and the thimble. 3. Read the value on the thimble corresponding to the intersection with the horizontal line on the sleeve. Reading on sleeve: 10000 µm No additional mark visible:...
  • Page 44 Danish Myo Technology A/S DMT-USA,Inc. E-mail: sales@dmt.dk E-mail: sales@dmt-usa.com Tel.: +45 87 41 11 00 Tel.: +1 734 707 0250 Fax: +45 87 41 11 01 Fax: +1 678 302 7013...

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