The Jackson Laboratory

3.0 Setup:

3.1 Reagent Preparation: Using the DOS computer, check the number of runs available for each reagent cartridge using the following sequence: from the Main Menu, press <F2> (“REAGENT LOAD”), and then <F5> (“REAGENT STATUS”). Next, page down through the reagents to check the number of runs left in each cartridge (under “AVAIL”) for the four desired chemistries. Change the reagent cartridges as necessary, using the following procedure: from the Main Menu, press <F2> (“REAGENT LOAD”), and then page down through the reagents until the cursor is on the reagent that needs changing. Press <SELECT> to highlight it, and then press <F1> (“AUTO LOAD”). When prompted to do so by the CX5, open the reagent door and remove the empty cartridge, taking care to slide it past the laser bar code reader (listen for the “beep”). Obtain the new reagent cartridge in the adjacent refrigerator, and load it past the bar code reader in the same manner into the CX5 when prompted to do so, after which the CX5 will check the levels in the new cartridge. After loading a new reagent cartridge, you must calibrate the CX5 for that reagent. Refer to calibration procedures below.

3.2 Calibration: From the Main Menu, press <F3> (“ CAL”), and then page down through the reagents until the cursor is over the reagent that needs to be calibrated. Press <SELECT> to highlight it, and then press <F1> (“CAL CUP ASSIGNMENT”). Enter the number of an available sector when prompted, and press <ENTER>. Place a single 0.5 ml sector cup in the first position (“cell”) of the previously assigned sector. Using the appropriate calibration reagent (described above in Section 2.3), squeeze several drops from the bottle into the sector cup, place the sector into the CX5, and press the green <START> button on the keyboard.

3.3 Control Preparation: Controls are run using the same procedure used for samples described below. The Lipid Controls (Levels 1 and 2) used for HDLc must be reacted with the HDL Cholesterol Separation Reagent, spun down, and extracted in the same manner, whereas the Comprehensive Chemistry Control Serum Levels 1, 2 and 3 are used directly.

3.4 Sample Preparation: The chemistry tests are run on mouse plasma which has been extracted from mouse blood in the laboratory. The mice are fasted for four hours, beginning one hour after the start of their light cycle, prior to the collection of the blood for the chemistry tests. At the conclusion of the fast, approximately 225 m l of blood (6-8 drops plus one full capillary tube) are collected from each mouse (depending on its size) using a retro-orbital bleed. The blood is collected into previously labeled 1.5 ml Eppendorf tubes containing 7.5µl of Heparin 1000u/ml and put on ice immediately. It is then spun down in a microcentrifuge at 14,000 rpm for 5 minutes. A minimum of 100µl of translucent blood plasma is collected from the upper layer using a 200µl Pipetman, leaving the blood cells behind in the Eppendorf tube which is discarded. This plasma is pipetted into a previously labeled 0.5ml Eppendorf tube and frozen for future analysis.

3.5 HDL Cholesterol : [Procedure used by the Heart, Lung, Blood & Sleep Disorders Center at The Jackson Laboratory differs slightly from the Beckman protocol]. Note—The following procedure uses six sectors (42 samples) as an example run which is a convenient size to run at a single time. If more or fewer samples need to be run, modify the procedure appropriately.

1. Arrange 42 empty unmarked 1.5ml Eppendorf tubes in a 96-place rack. Orient the rack as 12 rows x 8 columns, and skip every other row, placing seven tubes per row.

2. Record the plasma identifying information (mouse I.D. number) on a data sheet (lined paper), and assign a Beckman Coulter sector and cell number to each sample as in the following example. Be sure that the sector numbers assigned are actually available for imminent use.

3. Using a 250 m l repeating pipettor, add 35 m l of PEG (polyethylene glycol) to each open tube. Set the repeater for 35 m l, to multi-dispense 7 times, which will load 245 m l and fill one row of tubes (one sector's worth). Repeat this to complete the addition of PEG to the full rack. Using a 100 m l Pipetman, put 35µl of plasma into the bottom of each tube. Change tips between each plasma sample. Close the caps.

4. Label the caps with sector and cell number, i.e. 1-1 to 1-7 for Row One, 2-1 to 2-7 for Row Two, up through 6-1 to 6-7 for Row Six.

5. Vortex each Eppendorf tube for 10-15 seconds to mix solutions.

6. Let tubes sit for 15 minutes at room temperature.

7. Place the tubes sequentially into a refrigerated microcentrifuge set at 4 ° C with the hinges pointing out. Centrifuge for 10 minutes at 14,000 rpm.

8. Carefully remove the tubes from the centrifuge and replace them in the rack in the same six rows from which they were removed.

9. Locate the six Beckman Coulter amber-colored sectors previously assigned on the data sheet, and place seven new empty 0.5ml Beckman Coulter Microtube™ Tubecups into each sector, one in each cup holder.

10. Each tube should have in the bottom a white pellet containing the undesired precipitate. Carefully draw off the supernatant, avoiding contact with the pellet, and transfer to the corresponding sector cup. Discard the tubes containing the pellets.

11. It is important to avoid introducing air bubbles into the sample, as these will interfere with the results or cause the CX5 to be unable to detect the sample. To minimize introduction of air bubbles into the sample, carefully position the pipette tip at the bottom of the sector cup and raise the tip up the side of the sector cup as the plunger is being depressed, taking care not to inject air into the sample once all the liquid has been expelled. Often one air bubble will remain in spite of these precautions—this can be popped using the wooden sticks that come with the CX5 or a toothpick.

12. CAUTION: THE CUPS IN EACH SECTOR ARE NUMBERED FROM RIGHT TO LEFT. BE SURE TO LOAD THE SAMPLES INTO THE CORRECTLY NUMBERED SECTOR CUPS.

13. Occasionally, the precipitate will not form a pellet at the bottom of the tube, or the pellet will break apart easily. In these cases, there will be opaque particles floating on or suspended in the plasma sample. The presence of these particles will often cause the HDL result to be suppressed by the Beckman Coulter CX5. To avoid this problem, try to position the pipette tip in such a way as to draw up only clear plasma, leaving the particles behind in the Eppendorf tube. Even when this procedure is attempted, it can still be quite difficult in certain cases to successfully separate the plasma from the suspended particles.

3.6 Cholesterol, Glucose and Triglycerides:

1. The levels of these three components are measured directly from the blood plasma without further handling of plasma as is required for HDL.

2. Record the plasma identifying information (mouse I.D. number) on a data sheet (lined paper), and assign a Beckman Coulter sector and cell number to each sample as in the above example. Be sure that the sector numbers assigned are actually available for imminent use.

3. Locate the six Beckman Coulter amber-colored sectors previously assigned on the data sheet, and place seven new empty 0.5ml Beckman Coulter Microtube™ Tubecups (sector cups) into each sector, one in each cup holder.

4. Using a 100 m l Pipetman, pipette 35 m l of .9NS into the bottom of each previously assigned Tubecup. Then add 35 m l of plasma into each assigned cup. Change tips between each sample.

5. Follow suggestions in Subsection 3.5, #11 to avoid introduction of air bubbles into the sample.

6. CAUTION: THE CUPS IN EACH SECTOR ARE NUMBERED FROM RIGHT TO LEFT. BE SURE TO LOAD THE SAMPLES INTO THE CORRECTLY NUMBERED SECTOR CUPS.

4.0 Run:

4.1 The four chemistry tests used in the HLB PGA protocol cannot be run together and must be divided into two groups. The HDL test must be run separately because it utilizes a reagent to separate the two types of cholesterol from one another. The CHOL, GLU, and Tg tests can be run at the same time.

4.2 The tests are run in the following order: 1) HDL, 2) CHOL & GLU (labelled as a CHOL level), 3) TG.

4.3 The plasma used for the HDL test cannot be diluted but is calculated as a dilution factor of 2 because of the 35 m l PEG and 35 m l plasma. This should be removed first. If there is not enough plasma to run the other three tests, the plasma may be diluted in a 1:1 or 1:2 ratio.

4.4 Using the DOS computer, be sure there are enough runs left in the reagent cartridge for the number of samples to be tested. Follow the procedure described in Section 3.1, Reagent Preparation. This is maintained by the Diagnostic Lab Staff.

4.5 Program the CX5 to test the samples using the following procedure. From the Main Menu, press <F1> (“SAMPLE PROGRAM”), then <F2> (“PROGRAM BATCH”). Next, enter the sector numbers to program (e.g. “1-6”) where prompted, and press <ENTER>. The computer will respond with “Batch mode activated, (42) cups possible.” Enter number of cups in batch (e.g. “42”) where prompted, and press <ENTER>. Select the desired chemistries by scrolling through the list of possibilities, and press <SELECT> appropriately. Press <F8> (“END BATCH”) to program the CX5. “PLEASE WAIT -- WRITING TO DISK” will be displayed as the information is recorded.

4.6 If the status of the CX5 is “WAITING,” press the green <START> button on the keyboard to begin testing the samples.

4.7 If the CX5 is already operating, press the “LOAD” button on the CX5 itself to load the samples.

4.8 Load three of the programmed sectors into the CX5 and wait while they are automatically loaded onto the “Sample Wheel.” Load new sectors onto the wheel as space becomes available and remove sectors from the machine as the testing of those sectors is completed. It is important to monitor the progress of the CX5 closely in order to promptly troubleshoot any problems arising.

4.9 Clear the sectors from the computer so that they may be re-programmed for future tests. From the Main Menu, press <F1> (“SAMPLE PROGRAM”), then <F5> (“CLEAR SECTORS”), and then enter the sector numbers to clear (e.g. “1-6”) when prompted.

4.10 Remove and discard the sector cups once all desired tests have been run.

5.0 Clean Up: Discard the Eppendorf tubes, pipette tips, sector cups, and reagent cartridges into biohazard waste containers. Clean up any spilled liquids.

6.0 Data Reduction

6.1 The results will be printed on the dedicated dot-matrix printer as the CX5 finishes the test(s) per sample. Carefully monitor the activity of the printer to ensure uninterrupted feeding of the continuous-feed paper, and to collect the printed results in an accordion-folded stack, which will prevent the output paper from getting dragged into the printer with the paper supply, causing the printer to shut down. If this does happen, the results can be re-printed only up until the time when the sectors containing that data are “cleared” as described above in Section 4.9.

6.2 An electronic copy of the data will be sent at the same time it is printed to the Windows-based computer, and stored in the following folder: D:\Dawning Files\results. The files should be transferred as soon as the run is finished into the following folder within that one: D:\Dawning Files\results\H L B S\[test]\[set#], where [test] equals “HDL Cholesterol,” “Cholesterol & Glucose,” or “Triglycerides,” and [set#] equals “4 th set,” “5 th set,” etc. One folder should be used for each “run” where a “run” is defined as the maximum number of files for which tests were run sequentially and did not re-use the same numbered sector. Thus, once an individual sector is re-used for more samples (whether they are the same test or not), those files should be stored in a new folder as part of a different “run.” One the run is completed, its files should be condensed into a Zip file labeled “4 th_set.zip,” “5 th_set.zip,” etc. A copy of each zip file should be stored both in the folder labeled “zipped files” and on a 3.5”disk to be transferred to the data analyst.

7.0 Safety: No known safety concerns.

8.0 Time and Capacity: (The following is a rough estimate of time required for one technician to process 80 samples, although the work is much more efficient if several people work together for some of the tasks.)

9.0 Protocols: Mutagenized mice are bled for these chemistries at 8 weeks of age after consuming a regular chow diet, and again at 13 weeks of age after consuming a high fat, high cholesterol “atherogenic” diet.

10.0 Notes: Be sure to plan the sequence and timing of all tasks carefully. There are numerous procedures within this protocol that may not be stopped in the middle, such as the HDL Cholesterol sample preparation, followed immediately by the HDL test. The reagent cartridges in the CX5 need to be changed regularly which interrupts the run. The cartridge needs to be changed, calibrations done, controls run, and then the run of samples can be resumed. Plan well and good luck!