Atherosclerosis Risk in Communities Carotid MRI Study Manual 6 Flow Cytometry Laboratory

Transcription

1 Atherosclerosis Risk in Communities Carotid MRI Study Manual 6 Flow Cytometry Laboratory Prepared by the ARIC Carotid MRI Study Investigators For Copies, Please Contact ARIC Coordinating Center Department of Biostatistics (CSCC) University of North Carolina CB# 8030, Suite 203, Bank of America Building 137 E. Franklin Street Chapel Hill, NC Revised 02/15/05 MOP 3A: ARIC Carotid MRI, Retinal Photography Version 1.0

2 Manual 6: Flow Cytometry Laboratory Table of Contents 1.0 SAMPLE COLLECTION Sample shipping Sample receiving Sample preparation Reagents/supplies/equipment: Antibodies: Isotypic Controls: QC reagents: Equipment: Procedure Direct 3-color surface markers staining Monocytes Direct 2-color surface markers staining Platelets Combined direct surface CD14 and intracellular (COX-2; MPO) staining FLOW CYTOMETRY Flow cytometer start-up: DATA ACQUISITION QC samples Flow-Check fluorosphere (daily) Flow-Set fluorosphere (daily) Compensation procedure (weekly) Color protocol compensation procedure (FITC/PE): Color protocol compensation procedure (FITC/PE/PC5) Blood samples color protocols (FITC/PE): Color Protocols (FITC/PE/PC5) Cleaning Procedure Routine Cleaning Procedure Vacuum Line Cleaning Procedure Shutdown Procedure DATA MANAGEMENT /12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 2

3 1.0 SAMPLE COLLECTION 1.1 Sample drawing Venous blood is drawn into Cyto-Chex BCT vacutainer tube (Streck Laboratories, Omaha, NE, Cat. No ), according to Manual 2 instructions. Note: Cyto-Chex BCT tubes should be maintained at room temperature 18º to 30º C until use, up to the expiration date listed on the vial. Cyto-Chex BCT tubes should not be frozen. 1.2 Sample shipping BCT blood samples are shipped to the ARIC Flow Cytometry laboratory by an overnight courier on cold packs (making sure there is no direct contact between tubes and cold packs) to arrive the next morning at 10:00AM, and are processed immediately. 1.3 Sample receiving Log individual samples into flow cytometry lab shipping/arrival form via barcode scanning and complete the following log in information: date/time of arrival, name of laboratory technician, number of samples received, comments about the sample and batch condition, using pre-established codes. After checking the samples, make 2 copies of shipping/receiving forms for lipid and DNA lab and call them to pick up samples. Empty shipping boxes are packed and shipped back to the corresponding field centers, by DHL currier Instructions: go to login, and search the Receiver ID: ship to the receiver. Print out the bill, pack the boxes and schedule the pick up time with DHL. 1.4 Sample preparation Reagents/supplies/equipment: Pre-printed test labels with test names. Precision adjustable micropipettes (1-10ul; ul, ul, ul). Plastic disposable transfer pipettes. Polypropylene blue test tubes (12x75 mm) (VWR, Cat. No ) ImmunoPrep Reagent System (Beckman Coulter, Cat. No ) IntraPrep Permeabilization Reagent (Immunotech Coulter, Cat. No. IM2389) FBS (PBS with 2% FBS and with 0.1% sodium azide). 0.5% formaldehyde in FBS Antibodies: Conjugated monoclonal (mouse anti-human) antibodies: Beckman Coulter: CD3 PE (Cat. No. IM1282 ); CD14-FITC (Cat. No. IM0645); CD14- PC5 (Cat. No. IM2640); CD41-FITC (Cat. No. IM0649); CD45PC5 (Cat. No. IM2653); 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 3

4 CD61-FITC (Cat. No. IM1758); CD62P-PE (Cat. No. IM1759); CD 154-PE (Cat. No. IM2216); MPO-FITC (Myeloperoxidase) (Cat. No. IM1874). BD PharMingen: CD162-PE (PSGL-1) (Cat. No ) ebioscience: TLR-2-FITC (Cat. No ), TLR-4-PE (Cat. No ) CaymanChemical: COX-2-FITC (Cat. No ) Isotypic Controls: Conjugated (goat-antimouse) isotypes (IgG1-FITC, IgG2a-FITC, IgG1-PE, IgG2a-PE, IgG2a-PC5) QC reagents: Flow-Check fluorosphere (Beckman Coulter, Cat. No ) Flow-Set fluorosphere (Beckman Coulter, Cat. No ) Coulter Immuno-Trol control cells (Beckman Coulter, Cat. No ) Equipment: Beckman Coulter TQ-Prep and PrepPlus 2 Workstations Beckman Coulter EPICS XL Flow cytometer. Barcode scanner. Centrifuge with cooling system. Vortex mixer. 1.5 Procedure Label an appropriate number of blue test tubes (12x75mm) per participant with labels matching the original participant s ID and with a test name. Label an appropriate control tubes for each participant. Setup the TQ-Prep and PrepPlus 2 Workstations (start-up procedure). 1.6 Direct 3-color surface markers staining (Protocols: TLR-2FITC-C162PE-14PC5; 41FITC-TLR-4PE-14PC5) Monocytes Whole blood monocytes are stained with total of 6 conjugated monoclonal antibodies to surface markers; three antibodies are combined into one protocol. This step is done using Beckman Coulter TQ-Prep and PrepPlus 2 Workstation Mix each blood sample vial thoroughly by hand inversion 20 times and load into workstation carousel. Place all reagents into appropriate rack position in PrepPlus 2 workstation. Label 4 daughter tubes per each participant and place in carousel in PrepPlus 2 workstation. (Label 1: ID+IgG2aFITC-IgG1PE-IgG2aPC5; label 2: ID+IgG1FITC-IgG2aPE-IgG2aPC5; label 3: ID+TLR2FITC-162PE-14PC5; label 4: 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 4

5 ID+41FITC-TLR-4PE-14PC5).100ul aliquots of each blood sample are pipetted automatically into each labeled daughter tubes; Tube 1: 20ul of each isotype control conjugated IgG2aFITC and IgG1PE and 10ul of IgG2aPC5; Tube 2: 20ul of each isotype control conjugated IgG1FITC and IgG2aPE and 10ul of IgG2aPC5; Tube 3: 20 ul of each conjugated monoclonal antibodies TLR-2 FITC and CD162PE and 10ul of CD14PC5; Tube 4: 20 ul of each conjugated monoclonal antibodies CD41FITC and TLR-4PE and 10ul of CD14PC5 are pipetted into fourth tube. Samples are mixed and incubated for 20 min at RT in TQ-Prep. After that, a Reagent A is added, which lyses the red cells, into the first tube, and after that Reagent B is added, which stabilizes leukocytes, and then Reagent C, a cell membrane fixative. The same procedure is repeated for other tubes. Proceed to flow cytometry analysis. Note: A tube containing unstained patient blood cells may be used in addition to the isotype control and may be of value in evaluating autofluorescent vs. nonspecific binding properties of monoclonal antibody in individual patient specimens. This tube contains 50ul of FBS and 100ul of the participant s whole blood. Tube is put into TQ- Prep and processed as described above. Note: The specimens should be analyzed within 2h of the preparation when stored at RT. Otherwise, prepared samples should be stored at 2-8ºC in the dark and analyzed within 24h. 1.7 Direct 2-color surface markers staining (Protocols: CD61FITC-CD62PPE; CD41FITC-CD154PE) Platelets Blood sample staining for platelet analysis is done manually. Prepare total of three labeled daughter tubes per each participant ID for platelet analysis. Label 1: ID+IgG1FITC-IgG1PE; Label 2: ID+61FITC-62PPE; label 3: ID+41FITC-154PE. Pipett 47.5ul of FBS, 10ul of IgG1-FITC and 10ul of IgG1-PE, and 2.5ul of blood into first daughter tube; pipett 47.5ul of FBS, 10ul of CD61-FITC and 10ul of CD62P-PE, and 2.5ul of blood into second daughter tube; pipett 47.5ul of FBS, 10ul of CD41-FITC and 10ul of CD154-PE, and 2.5ul of blood into third daughter tube. Mix gently, incubate 15 min at room temperature in the dark, dilute with 1ml of FBS and proceed to flow cytometry analysis. 1.8 Combined direct surface CD14 and intracellular (COX-2; MPO) staining (Protocols: COX-2FITC-14PC5; MPO-FITC-45PC5) Blood samples are stained first with CD14-PC5 and CD45-PC5 and then they are subjected to the intracellular staining procedure. 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 5

6 Label 3 daughter tubes per each participant. Label 1: ID+IgG2aPC5-IgG1FITC; label 2: ID+14PC5-COX-2FITC; label 3: ID+45PC5-MPO-FITC. Add 10ul of IgG2aPC5 into tube one and 10 ul of CD14PC5 into second tube and 10ul of CD45PC5 into third tube. Dispense 50ul of whole blood into each daughter tube. Gently vortex tube by tube. Incubate for 15 min at room temperature in the dark. Add 100ul of IntraPrep Reagent 1 to each tube. Vigorously vortex tube by tube. Incubate for 15 min at room temperature in the dark. Wash each sample with 4ml FBS. Gently vortex. Centrifuge for 5 min at 300xg at RT Discard supernatant (by aspiration). Be careful not to disturb the pellet. Add 100ul of IntraPrep Reagent 2 (permeabilizing reagent) to each tube. Let mixing occur without vortexing. Incubate for 5 min at RT without vortexing. Gently agitate (manually), for 1-2 sec. Add 8ul of intracellular monoclonal conjugated COX-2-FITC antibody to tube two; 20ul of monoclonal conjugated MPO-FITC into third tube and 20 ul of the appropriate intracellular conjugated isotypic control (IgG1-FITC) to each control tube. Gently vortex tube by tube. Incubate for 60 min at RT in the dark. Add 4ml of FBS. Centrifuge for 5 min at 300xg at RT. Repeat wash procedure. Discard supernatant (by aspiration). Be careful not to disturb the pellet. Resuspend cells in 500ul of FBS containing 0.5% formaldehyde and proceed to flow cytometry analysis. Note: The specimens should be analyzed within 2h of the IntraPrep treatment when stored at RT. Otherwise, fixed samples should be stored at 2-8ºC in the dark and analyzed within 24h. 2.0 FLOW CYTOMETRY 2.1 Flow cytometer start-up: Power on the computer at least 30 min before reading samples. From the Windows desktop select (double click) EXPO32 ADC XL 4 color icon. Allow about 30 min to warm-up the system. 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 6

7 Verify the Power Supply Controls and Indicators: Open the Power Supply doors. Check the WATER TRAP, AIR FILTER, and VACuum FILTER. Check the SYStem VACuum gauge. It should read more than 17 in Hg. Check the SYStem PRESSure gauge. It should read between 28 and 32 psi. Check the VACuum trap. If it is more than ¼ full of fluid, clean it as explained in the Special Procedures and Troubleshooting manual. Close the power supply door. 3.0 DATA ACQUISITION 3.1 QC samples Flow-Check fluorosphere (daily) Flow-Check Fluorospheres consist of 10um polystyrene fluorescent microspheres suspended in an aqueous medium at 1x10 6 fluorospheres/ml. The fluorescence emission of the dye contained within the fluorospheres ranges from 525 nm to 700 nm when excited at 488 nm. They are used to verify instrument optical alignment and fluidics. Under Resource Explorer select: Protocols: Flow-Check. Check the Cytometer status message. Start this procedure when the Cytometer status message Awaiting sample appears. Vigorously mix the Flow-Set fluorosphere vial until no sediment remains at the bottom of the vial. Dispense drops of Flow-Check fluorospheres in a test tube. (Follow the package insert instructions for mixing and handling these fluorospheres). Put the tube on the sample stage. After data acquisition starts, look at the histogram 1 (FS/SS): ensure that the region includes only the main population in the center of the histogram. Edit the region if it does not fully enclose the population. All additional histograms are to be gated on the dense population of fluorospheres viewed on the FS/SS data plot. If necessary adjust the detector settings for the linear fluorescence signals such that each peak is approximately mid-scale on the histogram. Wait for data to be acquired on 5,000 events and acquisition to stop. Remove the tube. Record the HPCV (half-peak coefficient of variation) and peak position for the FS and each of the appropriate fluorescence parameters in a log. Collect at least 20 measurements (data points), at various time intervals following instrument warm up. Calculate and record the mean and the standard deviation (SD) of the HPCV and peak 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 7

8 position for each parameter. These values represent the expected range for the laboratory. Create a Levey-Jennings plot for the HPCV of each desired parameter using the mean and +/- 2SD of calculated range. Daily verification: Record the daily HPCV values for each parameter on its respective Levey-Jennings graph/table. 95% of values should fall within the +/- 2SD range for each parameter. If values are outside this range, prime the instrument and repeat. Print results out. Publish results to QC Flow-Check (lot #) Excel file. Expected results: Generally, a HPCV of <3% is acceptable for surface marker applications Flow-Set fluorosphere (daily) Flow-Set fluorospheres consist of 3.6um polystyrene fluorosphere suspended in an aqueous medium at 1x10 6 fluorospheres/ml. They are used to standardize three types of flow cytometer parameters: a) light scatter intensity (FS, SS or SS log); b) fluorescence intensity and c) hydrodynamic focusing (HPCV=half-peak coefficient of variance). Since the daily standard results are compared to reference values for the standard, it is important to establish these reference values. Select The Flow-Set protocol. Vigorously mix the Flow-Set fluorosphere vial until no sediment remains at the bottom of the vial. Dispense drops of Flow-Set fluorospheres in a test tube and load onto the sample stage. After data acquisition starts: look at histogram 1: ensure that the peak is within the region. If the PK position is not within the region, adjust the FS voltage as needed to place the population within the acceptance limits. Wait for data to be acquired on 10,000 events and acquisition to stop. Check that the peak position for each parameter is within acceptance limits and record them in a logbook. If the peak positions are not within the acceptance limits, adjust PMT high voltage to position the peaks within the acceptance limits Compensation procedure (weekly) Compensation procedure will be done weekly, for both 2-color and 3-color protocols. Immuno-Trol cells stained with mutually exclusive cell surface markers are used for fluorescence compensation adjustment. Immuno-Trol cells are stained with CD14FITC, CD14PC5, CD3PE and CD45PC5 antibodies and corresponding isotypic controls Color protocol compensation procedure (FITC/PE): Open 2-Color Protocol (FITC/PE) and select Setup mode from the cytometer control dialog. Ensure Baseline Offset is OFF. Place the isotypic control sample (IgG FITC/ IgG PE) on the sample stage. 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 8

9 Select the Detectors tab from the cytometer control dialog. Select linear amplification for forward scatter (FS) and side scatter (SS) gains. Adjust FS/SS gains and voltages to position cell populations as desired, within appropriately labeled (Ly, Mo, Gr) polygonal regions ( gates ) On the FL1 log vs. FL2 log dot plot, adjust FL1 and FL2 high voltages to extend the control data to about the end of the first decade. The percentages should be about 98% in the lower left quadrant on the dot plot and 1-2% in the regions on the histogram plots. Observe that the single parameter FL1log and FL2 log histograms demonstrate the negative population correspondingly. Adjust regions if necessary. Deselect the Setup mode from the cytometer control dialog and let the acquisition complete. Replace the isotypic control sample with the COMP sample labeled with specific FITCand PE- conjugated antibodies, to be used for compensation (CD14 FITC/CD2 PE). Adjust FS/SS gains and voltages to position cell populations as desired, within appropriately labeled (Ly, Mo, Gr) regions ( gates ), if necessary. Select the Compensation tab from the cytometer control dialog. Focusing on the FL2 log vs. FL1 log dot plot, adjust FL2-%FL1 compensation values and the FL1-%FL2 compensation value until the populations are positioned in the quadrants they belong. Deselect Setup Mode in the cytometer control dialog. Select Restart and allow acquisition to stop at the prescribed stop count. Verify the statistics on the FL1 log vs. Fl2 log dot plot; compensation is correct when the X-mean values for region A1 and A3 are within 0.1 of one another and the Y-mean values for region A3 and A4 are within 0.1 of one another. If the statistics show that compensation is not correct, repeat the previous 5 steps. Allow acquisition to stop at the prescribed stop count. Acquire a minimum of 2000 events for monocytes, events for platelets. Save data as LMD file and print out Color protocol compensation procedure (FITC/PE/PC5) Open 3-color protocol and select Setup mode from the cytometer control dialog. Ensure Baseline Offset is OFF. Place the isotypic control sample (IgG FITC/IgG PE/IgG PC5) on the sample stage. Adjust FS/SS gains and voltages to position cell populations as desired, within appropriately labeled (Ly, Mo, Gr) polygonal regions ( gates ) On the FL1 log vs. FL2 log dot plot, adjust FL1 and FL2 high voltages to extend the control data to about the end of the first decade. The percentages should be about 98% in the lower left quadrant on the dot plot and 1-2% in the regions on the histogram plots. 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 9

10 Repeat the previous step on the remaining fluorescence dot plots FL2 vs. FL4 and FL1/FL4). Observe that the single parameter histograms demonstrate the negative population correspondingly filling the first decade. Set gating and regions. Replace the isotypic control sample with the blood sample labeled with specific antibodies to be used for compensation. The following tubes are used to adjust the 3- color fluorescence compensation: COMP 1 (CD14FITC/CD2PE) COMP 2 (CD2PE/CD14PC5) COMP 3 (CD14FITC/CD2PE/CD45PC5) (VERIFY) Select Setup Mode on the Cytometer Control Dialog. Place the COMP 1 tube on the sample stage. Select Compensation tab from the Cytometer Control dialog. Focusing on the FL2 log vs. FL1 log dot plot, adjust FL2-%FL1 compensation values and the FL1-%FL2 compensation value until the populations are positioned in the quadrants they belong. View the statistics for the FL2 log vs. FL1 log dot plot. Compensation is correct if the mean X values for quadrants A1 and A3 are within 0.1 of one another and the mean Y values for quadrants A3 and A4 are within 0.1 from one another. Select Pause, remove the COMP1 tube and then place the COMP2 tube onto the stage sample. Select setup mode and compensation tab from the cytometer control dialog. Focusing on the FL4 log vs. FL2 log dot plot, adjust the FL4-%FL2 compensation value and the FL2-%FL4 compensation value until the populations are positioned in the quadrants they belong. Compensation is correct if the main X value for quadrants B1 and B3 are within 0.1 of one another and the mean Y value for B3 and B4 are within 0.1 from one another. Repeat as necessary. Deselect the Setup mode and allow acquisition to complete at the prescribed stop. Place the COMP 3 (VERIFY) CD14FITC/CD2PE/CD45PC5 sample onto the sample stage. Allow acquisition to stop at the prescribed Stop Count. Save data as LMD file and print out. 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 10

11 3.2 Blood samples After cytometer has been standardized and verified with Flow-Check and Flow-Set fluorospheres, Compensation set of samples are read first, then Immuno-Trol blood samples, and then other blood samples are read using 2-color or 3-color protocols color protocols (FITC/PE): Platelets: CD41FITC/CD154PE CDCD61FITC/CD62PPE Monocytes: CD14PC5/COX-2FITC CD45PC5/MPOFITC Color Protocols (FITC/PE/PC5) Monocytes: TLR-2FITC/CD162PE/CD14PC5 CD41FITC/TLR-4PE/CD14PC5 3.3 Cleaning Procedure There are two levels of cleaning: routine cleaning followed by sample head cleaning and vacuum line cleaning Routine Cleaning Procedure Perform the routine and sample head cleaning daily, before the vacuum line cleaning procedure and shutdown the instrument. Prepare a fresh cleaning solution of 1 part of high-quality bleach (Clorox) and 9 parts of distilled water or IsoFlow sheath fluid (10% bleach). Put 2-3ml of the bleach solution in a test tube. Prepare 5 tubes with 2-3 ml of distilled water. At the cytometer settings select: Panel: Cleaning panel. Check that cytometer status message reads Awaiting sample. Put the test tube containing water on the sample stage. When the sample stage lowers, remove the tube. Put the test tube containing the 2-3 ml of bleach solution on the sample stage. When the sample stage lowers, remove the tube. Run three tubes of distilled water or Iso-Flow sheath fluid. When the sample stage lowers, remove the tube. While wearing latex gloves, apply the 10% bleach solution to a gauze pad. Carefully push the moistened gauze pad up against the inside of the sample head and scrub away any debris inside and around the sample probe. Continue scrubbing the 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 11

12 sample head and probe by pushing the head up and down 10 times during a 60-second period. Replace gauze as needed. Rinse the sample head and probe with gauze moistened with water. Perform the Vacuum Line cleaning procedure Vacuum Line Cleaning Procedure Select the idle mode button. The RUN indicator on the cytometer will be flashing green. Fill a cleaning adaptor reservoir with 5 ml of distilled water. Install the adaptor just as a sample tube is installed on the sample stage. Wait for the water to be aspirated. Remove the adaptor and press the CLEANSE button. When the cleanse cycle ends, the CLEANSE button indicator turns off and the RUN button indicator flashes Shutdown Procedure After the routine cleaning and vacuum line cleaning procedures have been completed: Wipe down all exposed surfaces with 10% bleach solution followed by 70% ethanol. Place a test tube with DI water onto the sample stage. Exit from EXPO32 ADC software. Turn off the cytometer by selecting the XL OFF button from the Windows desktop. Reminder: Clean the air filter once a week. Clean the sheath fluid container once a month. Clean the cleaning agent container every 60 days. 4.0 DATA MANAGEMENT Print hard copies of each IDs flow result immediately after reading (daily) and put them into appropriately labeled binders. Export data from the flow LMD files into Excel (once a week/mondays). Verify that all IDs in the data output files match the sample database. Verify that all cell markers in the data output match the database. Transfer Excel data files to the CC by or by direct transfer to the CC server. 05/12/05 MOP 6: ARIC Carotid MRI, Flow Cytometry Laboratory Version 1.0 Page 12