SOP - Indirect Calorimetry --PUBLIC

Indirect Calorimetry Protocol (Chamber or Treadmill)

Purpose

To evaluate Metabolism via assessment of VO2, VCO2, RER, heat, and related measures under baseline or challenged conditions (ambient, thermoneutral, or heated temperature; at rest or with treadmill)

Experimental Design

Minimum number of animals: Recommend at least 5 animals per group being compared

Age at test: recommended at least 6 wks of age – younger may be possible if they are mobile

Sex: either

Time of Day: typically during the light phase, but may vary depending on study requirements

Equipment

Columbus Instruments OxyFast System

IDC chambers (for baseline or temperature challenge studies)

IDC compatible treadmill (for exercise challenge studies)

Warming chamber (for studies using elevated temperatures)

Procedure

Calibration (Typically performed by core personnel)

IDC must be calibrated every day it is used. The Door should remain shut during calibration procedures (to minimize changes in room air content, and to decrease noise in the core), but can be opened momentarily to leave the room as needed. # Turn the system on by flipping the switch on the power strip and take note of the time you first turned on the machine. For ‘Old’ IDC (8 chamber continuous monitoring system) make sure ALL necessary O2 AND CO2 sensors have been turned on.

1. Wait for the “Ready” light indicators on the CO2 sensors to turn on (should take about 15 min – you should be able to proceed with starting the PC and launching the software as the CO2 sensors initialize).
2. Power on PC. User name: administrator, Password: [Leave blank]
3. Launch “Oxymax for Windows”
4. Use default hardware configuration (110649 for old IDC)
5. Verify that all of the O2 sensors and CO2 sensors are reading “Ok” on the log. (Note, if only using a subset of the sensors – i.e. 2 sensors for treadmill or heat challenges, only the sensors that are on will show “Ok”, the sensors that are powered off may show an “Error” entry)
6. Click Tools->Calorimetry->Calibration to view O2 sensor reading.
7. Adjust the O2 sensor to read ~20.93 using the fine and course adjustment knobs on the O2 sensors.
8. Change fine knob to 5. Use coarse knob for adjustments for the first 2 hours, only use fine once values stop fluctuating that much
9. Repeat step #7 every 30 min until the machine has been turned on for at least 2-3 hours and the O2 sensor reading has “somewhat” stabilized. (i.e. O2 reading stays within ~0.25 of the set point) *30 mins for first 2 hours, then every 10-15 mins the last hour. Usually takes ~3 hours – longer/shorter depending on fluctuations.
10. Verify one in each pairing of dryer tubes still has new (blue) drierite. If not, empty out into old jar to be baked. Then fill with no more than an inch of new drierite at the bottom, and top off with recycled (bluish/white) drierite.
11. Attach calibration tube to calibration gas tank. But do not turn on tank!
12. Click “Start”
13. Watch the calibration sequence until it gets to the “Purging Span” steps, and then quickly turn on the calibration gas.
14. Wait for this step to complete, and you will be prompted to adjust the O2 analyzer to read ~20.55 for ‘New IDC’ and ~20.50 for ‘Old’ IDC. *Use fine knob for this step. Keep values close to +/- 0.05.
15. Adjust the knobs again until the numbers appear green and click ‘Next’
16. TURN OFF calibration gas and unhook tube when calibration is complete *Wait until gas pressure/regulator is 0 then unhook tube.

Protocol

(for IDC chambers)

1. Add a couple inches of BaSH water (can be poured from cage bottle) to each IDC water bottle to be used in experiment.
2. Weigh the water bottles, and record their weights on the IDC log. After weighing each bottle, place on counter in a line. Do not forget the order that they were placed in.
3. Place a chamber under each lid to be used. Make sure they are in the proper orientation.
4. Weigh/record 4 pellets of food and add to each chamber
5. Weigh/record mice one cage at a time, placing each mouse in its assigned chamber immediately after weighing.
6. Add water bottles to cages
7. File->Open experiment configuration->110649
8. Experiment->Setup->Browse->[go to pi folder and name file (recommend YYYY-MM-DD-PI-description)
9. Enter mouse IDs and weights. If running ‘Old’ IDC, uncheck the chambers that will not be used.
10. Turn on drier tubes and double check that all the fans are plugged in and running
11. Experiment->Run
12. Experiment will run for approximately 50 intervals for ‘New’ IDC and 242 for ‘Old’ IDC; (***Note this is for an overnight run, if shorter durations are needed, the interval settings can be adjusted***)
13. Experiment->Stop
14. File->Export->Export All Subject CSVs->Yes (Overwrite)
15. Turn off drier tubes
16. If this is the last round of IDC for the week, turn the power strip off
17. Remove one mouse at a time, record their weight, and place them back into their cage.
18. Weigh/record the food and water bottles. If you plan to begin another round of IDC, be sure to organize the water bottles so that the ending weight can be used to begin the next round.
19. Clean/dry cages

(for treadmill assay)

1. Depending on the study design you may wish to perform a couple of habituation sessions to the treadmill for the 2-3 days prior to the data collection date.
2. Confirm setting for the treadmill and shocker (Recommend starting speed @6, Shocker Repetition Rate @3, Intensity @9)
3. Preclean the treadmill using RTU disinfectant
4. Weigh/record mice one cage at a time, placing each mouse in its assigned chamber immediately after weighing.
5. File->Open experiment configuration->110649-treadmill.exp-lydia.exp
6. Experiment->Setup->Browse->[go to pi folder and name file (recommend YYYY-MM-DD-PI-description)
7. Enter mouse IDs and weights. If running ‘Old’ IDC, uncheck the chambers that will not be used.
8. Turn on drier tubes and double check that all the fans are plugged in and running
9. Place animal in treadmill (treadmill should be ‘stopped’ and ‘shocker’ should be ‘off’ when placing mouse – standard protocol will leave the treadmill off until after the 1^st data collection from the mouse has started)
10. Experiment->Run
11. Adjust treadmill speed as needed for study (recommend treadmill off during reference and 1^st sampling interval, then set @ speed of 6, and increase by 2 for every 2 minute interval) –
    1. you can use the add comments feature of the software to annotate the speeds and animal statuses
12. If animal is exhausted turn off shocker, and record the time to exhaustion(recommended criteria is 3 seconds of contact with the shocker, or 3 separate brief contacts with the shocker – can adjust as needed for your study, but should be consistent with your IACUC animal protocol)
13. Experiment->Stop
14. Remove animals from treadmill and clean treadmill with RTU disinfectant
15. File->Export->Export All Subject CSVs->Yes (Overwrite)
16. If repeating for another set of animals,
    1. Create a new experiment file “Experiment->Setup->Browse->[go to pi folder and name file (recommend YYYY-MM-DD-PI-description)”
    2. Repeat steps 7 - 15
17. Turn off drier tubes
18. Exit Software and Turn off computer
19. Turn off power strips
20. Turn off Treadmill Power Supply
21. Clean up the room

Data Outcome Measures Produced

Common parameters of interest

• VO₂ : Volume of O2 consumed by the animal

• VCO₂ : Volume of CO2 production by the animal

• RER : Respiratory Exchange Rate ... VCO₂/VO₂ ... reflects the energy source utilized by the animal (i.e. low RER approaching 0.7 indicates fatty acid oxidation, high RER approaching 1.0 indicates carbohydrate metabolism)

• Heat : Estimated energy produced by metabolism (typically expressed as Joules or Calories)