Buxco Bench Protocol
Machine Start-Up:
- Turn on computer, screen and sound (speaker).
- Turn on BUXCO nebulizer control-10, BUXCO Max II Strain-Gage Preamplifier and all BUXCO Bias Flow Regulators.
- Click BUXCO Nebulizer Control-10 to Computer Ctrl by pressing the MENU button (white) until the Computer Ctrl option comes up. The Trickle out should be at .8 and the Dilution out should be set at 10.
- Computer will prompt "Log on to Windows"; click OK.
- At computer main menu, double click on Biosystem XA.
- At Change Operator screen, click OK.
- Go to System ? calibrate where you will see toggle on Strain-Gauge Preamplifier for DC ? AC ? OFF. Turn all Preamps to DC.
- Click box 1 and be sure name next to number is also highlighted.
- Go to cell 1 and turn black lever on top of cell to right to close off flow at top of cell. Remove Cell 1 line from Bias flow regulator (located towards base of cell). Attach 1 ml syringe with tubing to Bias Flow port. Draw back 1 ml of air. Adjust measurement line to "0" by using the "zero" turn knob on the Preamps. If area to adjust is too large or unable to turn "zero" knob, use BUXCO supplied screw driver to balance screw and turn clockwise or counterclockwise to bring measurement line near "0".
- Inject 1 ml air bolus into cell with steady pressure. You will see the measurement line move as the air enters into the chamber. When the injection is done, the measurement line should return to zero line. You have 5 seconds to click F7 to freeze injection graph. Use mouse to move right side of red measuring line to close off injection graph. Click manual setting to receive reading of injection. It should read between +/- 1.8 to +/- 2.7. If you canít get these readings, use the gain to adjust your measurement. If reading is within range, move toggle on Preamps to AC. You may have to let it scroll 2 times for the line to return to 0. Then click F8 to re-zero cell.
- Click Next to move onto next cell. In order to receive reading from cell, you must highlight both the cell box and the cell name.
- When all 12 cells are calibrated, click save and close.
- Go to file ? open ? Hypercapnia.WXA ? click open. Computer will display all 12 cells with its measurement line in each box. Now itís time to balance cells.
- Place Preamps toggles back to DC. Starting with cell #1 and moving to cell #12 adjust the amount of LPM on the Bias Flow regulators so that the measurement line stays at 1.0. Then move on to cell 2, 3, etc. until all the cells are balanced at 1.0. Once you adjust one cell, the other levels may move up or down. Just adjust each with the LPM knob on regulator. Balance is complete when all cells are at 1.0. Flip all Preamps toggles to AC. Now youíre ready to measure the mice.
Running Subjects:
- Obtain 12 mice subjects and place into cells.
- Record database ID number on BUXCO worksheet.
- Allow mice to acclimate for 20 minutes. Meanwhile, click all boxes in PROTOCOL and RECORDING tables. In RECORDING screen, click RUN which allows you to enter the database number in the cells.
- In session description, enter date as mmddyy A or B (A for the first study and B for the second study).
- In study name enter date as mmddyy AS or BS (AS for first study and BS for second study).
- Click CONNECT to activate cells with computer.
- After completing acclimation time, click Site 1 PROTOCOL and view the protocol as it is running.
- Go to PROTOCOL table and click GO ON to start experiment on room air.
- Go back to Site 1 table to view task and description. Follow directions. Most tasks will beep when you need to do something with the gases or the methycholine (MC). However, task 47 PAUSE add Saline will not beep; keep an eye on computer during that time.
- During the recovery time from each gas, the hose should be hooked up to the nebulizer and the next gas turned on to fill the flask (bubbler) which should have a little water in the bottom of it. The tube from the flask wonít be connected to anything. The next gas you are waiting to run will be released into the room until the protocol tells you to start the next gas. Adjust the gas flow with the increase/decrease knob on the tank until it measures 9 liters on the flow meter.
- At the end of the experiment, the computer will beep. Go to RECORDING table and click STOP. Computer will prompt "Do you want to stop recording?"; click YES.
Data Collection: - Go to FILE ? EXPORT FROM DATABASE.
- Find date and study name.
- Click DATE.
- Click EXPORT.
- A calendar will be displayed.
- Click OK.
- Will see computer generating files for export.
- When complete, go to file ? exit.
- Double click on WINDOWS EXPLORER.
- Go to My Computer.
- Go to (C) local drive.
- Click on PROGRAM FILE ? BUXCO ? DATA.
- Click on date of study.
- Open study and click on EXPORT FILES; then close file and close data file.
- Highlight folder with desired date with left click and slide file over with right click to DRIVE E: COMPACT DISK.
- Remove right click.
- Computer will prompt MOVE HERE ? COPY HERE ? CANCEL.
- Click on COPY HERE.
It takes about 15 minutes to transfer 2 sets of BUXCO test data to the zip drive. While waiting for the data to transfer, clean up the cells. When the process is complete, exit out of Windows Explorer and turn off the computer and the BUXCO equipment.
Program for Genomic Applications (PGA)
Supported by the National Heart, Lung, and Blood Institute (NHLBI) (Grant # HL66611)
