Protocol Index

Comprehensive Cage Monitoring Systems (CCMS) Protocol

Prepared by: Stacey Dannenberg
Date Prepared: August 13, 2002
Code: MP-012
Reviewed by: RVS
Revision #: 1.0
Date Revised: April 12, 2004


0.0 Abstract:  The CCMS diagnostic instrument is manufactured by:
Columbus Instruments
950 North Hague Avenue
Columbus, OH 43204-2121
It is designed to study mouse movement and activity level, sleep patterns, and food and water consumption. It provides us with metabolic data, total food and drink consumption data, activity data, and the exchange of Oxygen and Carbon Dioxide (RER) data.
1.0 Instrument Layout: The CCMS diagnostic instrument has 16 individual mouse chambers. It is connected to a Win 2000 Dell computer. It has an O2 and C02 delivery and analysis system that is attached to the 16 chambers. Each chamber has individual Mettler Toledo scales and drinking tubes.
2.0 Reagents and Expendables:
Item: Vendor:
Magnesium Percholate Sigma
Iso Pads Harlan Teklad
Mettler Toledo Scales Columbus Instruments
Food Hoppers Columbus Instruments
Glass Dryer Column Columbus Instruments
Parts and Service Columbus Instruments
One tank of 0.5% Carbon Dioxide with
20.5% Oxygen mixed and balanced with Nitrogen 		Advantage Gases and Tools
6% (fat) meal (crushed Lab 6% chow) N/A
Liqui-Nox  cleanser  Alconox, Inc.
70% alcohol N/A
3.0 Setup:
3.1 Set up the clean cages on their pedestals. Place an Iso Pad, then place the metal floor grid. Alcohol the drinking tubes as well as the top of the lid. Close the cage lid. Check the water bottles to see if they need to be refilled. Fill the food container, set it in the scale, and attach it to the cage system. Double check the scale to make sure that it is not bouncing. If it is bouncing, adjust either the scale or the food container. Once that is adjusted, fill and attach the cylinders containing the Magnesium Perchlorate. This is good for 5 runs. To change a cylinder, take it into the fume hood, change it and dispose of it properly.  Gloves must be worn for this procedure.  Empty the old Magnesium Perchlorate into the original glass jar it came in and refill with a new jar. It is very important to wear gloves, as Magnesium Perchlorate may irritate the skin. Make sure that the Magnesium Percholate does not come in contact with any paper products. Magnesium Perchlorate is a strong oxidizer and may cause spontaneous combustion. It is water-soluble, but be aware that it does become very warm when dissolved in water.

3.2 Leak Test: Once set up is complete, perform a leak test. Two people are required to do this (one person at the computer and the other at the cages). The person working at the computer performs the following functions:

  • Open the Oxymax Program
  • Under tools, click on diagnostics
  • Click on sample pump button
  • Click the on button
  • Select chamber one
    While the person at the cages holds the cylinder red hose going to the Magnesium tube, the person at the computer will observe the red ball drop to zero and say ok. This procedure must be repeated for all of the sixteen cages.  Once this process is complete, the person at the computer should click exit. After this, label the mice that have been selected for the next run. This can be done the day before the actual run. You may also enter the mice into the computer and save it for the next day. This will save a lot of time. On the day of testing, the mice selected for the run are weighed in the CCMS cage room and put into the cages. As you weigh the animals record them on your work sheet so you can enter the data into the computer, as well as a record for yourself.

    3.3 Calibration: The C02 and 02 sensors must be calibrated. This calibration is accomplished by plugging into a tank of 0.5% Carbon Dioxide and 20.5% Oxygen which is balanced with Nitrogen. This must be a certified gas. To calibrate, return to the computer and perform the following functions:

  • Open the Oxymax Program
  • Click on file - open
  • Select standard HLB, or the appropriate project
  • Select the calibration tab (this should be in the lower left-hand side). If the air pump is on, switch it to off. Check the level of gas in the tank . If it is lower than 200 psi, it is time to reorder a new tank. It is a good idea however to have a back up tank on hand incase you have a leak, or something goes wrong with the tank or they sent you the wrong one.
  • Click on C02 offset first.Wait about 1 minute for it to reach a .000 reading. Make sure that it fluctuates between .001 and .000 to ensure that the value is not below 000.
  • Once that has been achieved, select C02 gain. It will take approximately 2 minutes for it to read, the value. It is usually between .499-.500. You will need to calibrate it to what the certified tag says on the tank tab on the gas cylinder. (See note) For example: 0.503. Adjust the dial labeled C02 if necessary to obtain this reading.
  • Click on 02. This must read 20.50, or again, the value listed on the certified tag. (See note)  Adjust as necessary with either the fine or coarse dials.
  • Once this process has been completed , exit the program and remove the hose from the tank of gas.
  • Note: The CO2 reading of .499-500 and the O2 reading of 20.50 can change. These values are determined by the exact readings as described in the certified letter which accompanies the tank.

    3.4 Experiment Configuration: After you have calibrated the system, click the experiment configuration tab. Enter the data file name under the “D drive” and the copy file name under the “D” drive. Click apply, save and ok. Select the chamber configuration tab. Click on chamber one and enter the mouse’s ID#, the weight, the sex, the born date, the experiment date, and the name of the technician (‘s) performing  the run. Click apply. If the next mouse’s information is the same, click duplicate to display the previous information. Then click apply again. Continue this procedure for the remaining chambers.

    • 4.0 Run: Once setup is complete, turn the air pump on and make sure that it reads 60. Now you may hit the run button. After clicking the run button, a message will appear asking if you wish to purge the system. Click yes. This will purge the water lines for the top 8 cages. Then a message will appear which says click ok when the system has finished. When you do not hear any more “clicks” from the water pump box, then click ok. Another message will appear again asking if you wish to purge the system. Click yes so that you can purge the water lines for the bottom 8 cages. Click ok when you do not hear any more “clicks”. The computer will then zero out the scales. It will not continue if a scale is unbalanced due to improper alignment or if there is a mouse in the food hopper. Once the problem is resolved, the computer will continue to finish checking the rest of the scales. This will take you to a data screen. During the run, check on the cages and computer information twice a day to make sure everything is ok and that there are no error messages.  Each run lasts for 72 hours. After 72 hours, you will need to wait for it to be at least one hour past the start time on the last cage, click stop to end the experiment.
    5.0 Clean Up: When the experiment is complete, click stop. Shut off the air and remove the mice. Weigh the mice before putting them back in their cages. Break the cages down by removing and dumping the left over food from the food hopper. Remove the metal floor grid and dump the Iso pad and any feces that may be in the cage. Take the grids, cages and food hoppers to the sink and wash everything in Liqui-nox. Next, dry the food hoppers in front of a fan for faster drying. The cages and metal floor grids can dry on the racks in the CCMS room. Once everything is dry, set it up again for the next run. The Magnesium Perchlorate must be changed after the 5th run and should be changed at this point if it is time to do so. Sterilize the drinking tubes by using a spray bottle of 70% alcohol and spraying the drinking tubes and the tops of the cages. Then wipe and let dry.
    6.0 Data Reduction: See Appendix in CCMS Protocol Word Document.
    7.0 Safety: When handling the Magnesium Perchlorate, you must wear gloves at all times. When disposing this substance, make sure that it doesn't come in contact with paper products since Magnesium Perchlorate is a strong oxidizer. Call the Safety Office and have them pick it up for proper disposal. Any spills should be wiped up with a sponge, and the sponge should be rinsed under water immediately. Magnesium Perchlorate is very water-soluble. If you should drop a jar of Magnesium Perchlorate sweep it up in a 1-quart sharps container and take it to the sink and rinse it under water through a strainer. This way it is out of the plastic container and the glass left behind. The other thing you could do is to sweep it into an empty jar if available.
    8.0 Time: It takes 72 hours for the experiment to run. It takes about 15 minutes to weigh the mice before and 15 minutes after the test. It takes 10 minutes to put the mice into the chambers.  It takes 10-15 minutes to calibrate the machine and 10-15 minutes to perform a leak test. Set up time with 1 person is 25 minutes. Clean up time with 2 people is about 35 minutes.
    9.0

    Protocol:

    Setup (can be performed the day before the run):
  • Select the mice you wish to use and tag them.
  • Put the cages together.
  • Fill the water bottles.
  • Perform the leak test.
    The day of the run:
  • Weigh the mice.
  • Perform the calibration.
  • Enter mouse ID and information into computer system.
  • Place the mice in the designated chambers.
  • Check the scales to make sure they are stable. Click run.
  • Purge system. Click ok.
  • Experiment will begin to run after it zeros the scales.
    During the run:
  • Check on the experiment twice a day for errors and to make sure that the mice are alive and healthy. Check the computer to confirm that the mice are able to drink and eat. Also, it is very important to check the temperature and humidity in the room as these factors can contribute to misleading data.
  • The experiment will run for 72 hours. Stop the experiment at least 72 hours after the start time. Example if you start at 8:00 am you will need to check cage 16 to make sure that it is 72 hours after 8:00 am. In reality it runs about 73 hours instead of the 72 hours.
  • Remove and weigh the mice.
  • Return mice to their proper cages.
  • Wash the cages.
  • Dry and setup for next run.
    • 10.0 Notes:

    Protocol Index

     

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    Program for Genomic Applications (PGA)

    Supported by the National Heart, Lung, and Blood Institute (NHLBI) (Grant # HL66611)