Using the BD LSRFortessa. Penn State Microscopy & Cytometry Facility Flow Cytometry lab

Transcription

1 Using the BD LSRFortessa Penn State Microscopy & Cytometry Facility Flow Cytometry lab

2 Table of Contents 1. Start-Up 2. Computer and Software Login 3. Setting Up Your Experiment 3.1. Setting Up the Workspace 3.2. Creating a New Experiment 4. Setting Up for Tube Mode Operation 4.1. Switch the Fluidics to Tube Mode 4.2. Creating a New Specimen 4.3. Creating a New Tube 5. Setting up for HTS (Plate) Mode Operation 5.1. Ensure connection is correct & HTS is powered ON 5.2. Creating a Plate 5.3. Setting up a Plate 6. Setting up Cytometer Settings and Graphs 6.1. Selecting Parameters 6.2. Selecting H as a Parameter 6.3. Creating a Dot Plot 6.4. Creating a Gate 6.5. Selecting Parameters for Dot Plot Axes 6.6. Creating an FSC-A vs. SSC-A Dot Plot 6.7. Gating 6.8. Opening an Experiment Layout 6.9. Labeling Parameters Setting Up the Number of Events to Record Setting Up Voltages Based on an Unstained Control Adjusting Area Scaling for FSC Adjusting Area Scaling for Each Laser Checking the Brightest Tubes Saving Application Settings Saving Experiment Template 7. Acquiring Compensation Controls and Calculating Compensation 7.1. Creating a Compensation Control Specimen 7.2. Recording Compensation Tube Data 7.3. Calculating Compensation 8. Running Samples 9. Exporting and Transferring Recorded Data 10. Cleaning after HTS Mode 11. Cleaning after Tube Mode 12. Batch Analysis 13. Logging Out from the Software 14. Shut-Down 2

3 1. Start-Up 1. Turn on the FACS Flow Supply Cart on the floor with the green button. 2. Turn on the cytometer with the green button. 3. Turn on the computer. 4. Log in to the computer. The login information is on the computer screen. 2. Computer and Software Login -To log into the computer, the user-name and password that is posted on the computer. -Open an internet browser and log-in to RIMS. -To log in to the FACSDiva software you need to locate the icon on the desktop and double click it. -In the login window, scroll down through the list of user names to locate yours. The format of the user name for FACSDiva software is the first letter of your first name followed by your full last name. If you forget your password, alert a staff member and it can be reset. 3

4 3. Setting Up Your Experiment 3.1. Setting Up the Workspace Before you start, make sure that you have all the necessary windows open. Go to the View menu and make sure the following boxes are checked: Browser Cytometer Inspector Worksheet Acquisition Dashboard Once the software finishes loading, the instrument should automatically connect to the software. In the Cytometer window, it should say Cytometer Connected. It might also say Fluidics not ready, which is OK just click Clear. If prompted about CST settings, choose Use CST Settings. 4

5 3.2. Creating a New Experiment To create a new experiment from scratch, click the New Experiment icon ( ) in the browser toolbar. The new experiment will be created with a generic name. Right click on the name of the experiment. Select Rename. Rename it using the date and specific information of your experiment to make your name unique. To create a new experiment from an existing template, from the top menu bar choose Experiment>New Experiment. Then find the template you wish to use. 4. Setting Up for Tube Mode Operation 4.1. Switch the Fluidics to Tube Mode Place the cytometer in Standby. Turn off the HTS using the power button on the back of the unit. Detach the sample coupler from the cytometer SIT by unscrewing the top brown thumbscrew. Once it is loose, pull straight down. Remove the SIT protector by unscrewing the gray conical nut. Slide the SIT protector straight down. Ensure that liquid drips from the SIT. 5

6 Install the DCM sleeve, which is attached to the side of the cytometer in a small tube. Place the SIT protector in this tube while in Tube Mode. To install the DCM sleeve, slide the sleeve straight up over the SIT and screw the conical nut on to secure it. Install a tube with no more than 1mL DI water on the SIT, then place the tube support arm under the tube. Switch the acquisition control on the side of the cytometer to Tube Mode: Press Prime. Ensure that bubbles appear in the tube Creating a New Specimen Click the New Specimen icon ( ) to add a specimen and a tube to the experiment; click once on the plus sign (+) next to Specimen_001 to expand it Creating a New Tube Click the New Tube icon ( ) to add second tube. Add as many tubes as you need. You do not need to add tubes that are compensation controls. In the browser, click the icon to the far left of the tube named Tube_001. The pointer changes to green. The current tube pointer indicates the tube to which instrument setting adjustments will apply and for which acquisition data will be shown. 6

7 5. Setting up for HTS (Plate) Mode Operation 5.1. Ensure connection is correct & HTS is powered ON The default cytometer setup is Plate Mode (HTS). Be sure to check that the device is powered on using the button on the back. Also ensure that the sample coupler thumbscrew is correctly attached to the SIT. Place the cytometer in Run, then run HTS>Prime. During the prime cycle, check for leaks on each of the 2 syringe pumps and also at the SIT Creating a Plate Use the New Plate button in the Browser toolbar to add a default plate, or choose one from a template using Experiment>New Plate Setting up a Plate In the Plate window, choose High or Standard Throughput. Select the well(s) of interest, and click the pink (Setup Control) or blue (Specimen) buttons to add wells. Highlight the well(s) of interest and adjust Loader Settings as desired for that group of wells. Repeat the process of selecting wells, choosing pink/blue wells, and adjusting Loader Settings, for all wells of interest. Do not add wells for compensation samples at this time. Wells can be renamed in the Inspector window under the Well tab. 7

8 6. Setting Up Cytometer Settings and Graphs 6.1. Selecting Parameters Click the Parameters tab in the Cytometer window and delete all the parameters that you don t need. To delete parameters, click the selection button next to each parameter that you want to delete. Hold down the Ctrl key to select more than one. After you are finished selecting, click the Delete button. You can add parameters by clicking the Add button. You can choose which parameters to use by using a scroll down menu: 8

9 6.2. Selecting H as a Parameter Check H for all parameters except SSC Creating a Dot Plot Create a FSC-A vs. FSC-H plot on the global worksheet. Select the Dot Plot icon ( ) on the Worksheet toolbar and click once in the upper left corner of the worksheet field. A default size plot is drawn Creating a Gate Create a gate by selecting the Polygonal Gate button and drawing it in the dot plot. Be sure you finish your gate at the same point you started drawing it. This gate will let you gate on a single cell population and exclude aggregates. This gating strategy may not be appropriate for your cell type. For example, If you are working with whole blood, bone marrow, or with samples that may contain more than one cell type, you may skip the gating step. 9

10 6.5. Selecting Parameters for Dot Plot Axes Create another dot plot next to the FSC-A vs. FSC-H dot plot. This time, make it FSC-A vs. SSC-A. Change the parameters on your dot plot by left clicking on them and selecting the one you need in the pop-up menu. To gate it on the P1 gate, right click inside the FSC-A vs. SSC-A dot plot and in the popup menu select Show Populations, then P Creating an FSC-A vs. SSC-A Dot Plot Create a gate on the FSC-A vs. SSC-A dot plot. Later you can adjust it to fit your population precisely. 10

11 6.7. Gating Create as many dot plots (2 parameters) or histograms (1 parameter) as you need to show all your fluorochrome combinations. Gate them all on the P2 gate the same way as you did on the P1 gate previously Opening an Experiment Layout Choose Experiment > Experiment Layout. ***For HTS mode, first select all the wells of interest from the Plate window!! 6.9. Labeling Parameters Define labels for each parameter. 2. Type Label Name 1. Select Parameter As an alternative, you may apply existed labels from the list on the right. 11

12 6.10. Setting Up the Number of Events to Record In the Experiment Layout dialog box, click the Acquisition tab. Here you can set the amount of events to record for each tube. If you don t have specific preferences, we recommend that you record the same number of events in every tube. Select all tubes by clicking on the selecting dots located to the far left of your tube names while holding Shift. Then specify the number of events by using the Events to Record scroll down menu. After that, select Global Worksheet. Then, select Stopping Gate. Leave Storage Gate value equal to All Events After everything is set, click the OK button in the right lower corner of the Experiment Layout window. 12

13 6.11. Setting Up Voltages Based on an Unstained/Negative Control Now you are ready to being acquiring your samples. Your first tube/well should be an unstained and untreated control. Make sure that the pointer on the far left from the tube name is activated, or that the well is selected. If using a tube, place the tube on the Sample Injection Port (SIP), or place the plate on the HTS. Press the Low and the Run buttons on the cytometer. For HTS mode, set this well s Loader Settings to have 0.5 as the Sample Flow Rate. For either mode, click the Acquire Data button on the Acquisition Dashboard. Adjust Voltages so you can see the population of interest in the center of the FSC-A vs. SSC-A dot plot. Adjust the voltages so the autofluorescent signals on each parameter form peaks within the first decade. Generally it is nice to be able to see both the ascending and descending slopes of each peak. 13

14 6.12. Adjusting Area Scaling for FSC Check area scaling for forward scatter: go to the Laser tab in the Cytometer window and adjust the FSC Area Scaling value until the population of interest on the FSC-A/FSC-H dot plot is aligned along the diagonal of the square dot plot box: Normal Decrease FSC Area Increase FSC Area Scaling for optimal Scaling for optimal settings settings 14

15 6.13. Adjusting Area Scaling for Each Laser Check area scaling for one parameter for each laser you are going to use in your experiment. Go to the Laser tab in the Cytometer window and adjust the Blue laser Area Scaling value until the population of interest on the Parameter-A vs. Parameter-H dot plot is aligned along the diagonal of the square dot plot box. In this example we are adjusting Area Scaling for the Blue laser using a FITC stained sample: Normal You need to decrease You need to increase Blue laser Area Scaling Blue laser Area Scaling 15

16 6.14. Checking the Brightest Tubes Check the brightest samples to insure all the data that you are going to collect will fall within the scale. After this step, the H parameter can be deselected for all fluorochromes Saving Application Settings Once you are satisfied with your settings, you can right-click on Cytometer Settings in the Browser and choose Application Settings > Save to save the settings for future use. Use the naming format: PI-Name_YourName_YourExperimentName Saving Experiment Template If you wish to create an Experiment Template using these settings, create a new Experiment using Experiment > New Experiment > Blank Experiment. Then, rightclick Cytometer Settings and choose Application Settings > Apply. Then, add any tubes/plates/wells that you d like to be part of the template. Finally, save by right-clicking on the name of the Experiment and choosing Export > Experiment Template. Use the naming format: PI-Name_YourName_YourExperimentName. 16

17 7. Acquiring Compensation Controls and Calculating Compensation 7.1. Creating a Compensation Control Specimen For Tube Mode: Create Compensation Controls if you are going to use more than one fluorochrome. Select Compensation Setup from the Experiment menu; then select Create Compensation Controls. For HTS Mode: Create Compensation Controls if you are going to use more than one fluorochrome. Click on the first well that contains your first compensation sample and choose Compensation Setup from the Experiment menu; then select Create Compensation Controls. Each fluorophore is associated with one or several labels. For the majority of applications you can use the default label, Generic, and delete the rest of the entries. If within the experiment you are using different labels for the same fluorochromes and you have corresponding compensation controls for them, then you may choose to create additional compensation control tubes. Use the Add and Delete buttons to modify your preferences. Click the OK button to proceed Recording Compensation Tube Data Global Worksheet should change to Normal Worksheet automatically. In Tube Mode, click once on the plus sign (+) next to the Compensation Controls Specimen to expand it. Select Unstained Control by clicking on the icon to the left of the tube name or the appropriate well. Place the tube with the unstained sample on the Sample Injection Port (SIP), if in Tube Mode. Press the Low and the Run buttons on the cytometer. Click the Acquire Data button, wait a few seconds until the threshold rate 17

18 is stable, and then click the Record Data button on the Acquisition Dashboard. Record data for all tubes in the Compensation Control Specimen. For HTS Mode, you can select all the wells of Compensation Controls and click Run Wells, or you can run each manually. After you are done recording, adjust the P1 gate of the FIRST sample s graph ONLY to include your population of interest. Then you may right click on the P1 gate and select Apply to All Compensation Controls. If this does not give the desired result, adjust P1 for each sample. Adjust P2 gates as needed for each sample Calculating Compensation Navigate to Experiment, then Compensation Setup, and then select Calculate Compensation. The computer will calculate compensation for all parameters. In the window that appears, select Link & Save 18

19 8. Running Samples Switch back to Global Worksheet by clicking on the top left icon. Now you are ready to collect data for your samples. The pointer on the far left of the tube for which you are about to record the data should be selected (Tube mode). Or, choose the wells you wish to record (HTS mode), either all at once using Run Plate or one at a time using Acquire and Record OR Run Well. If using a tube, place the tube on the Sample Injection Port (SIP), or place the plate on the HTS. Be sure the cytometer is in Run. For tubes, click the Acquire Data button, wait a few seconds until the threshold rate is stable, and then click the Record Data button on the Acquisition Dashboard. DO NOT FORGET TO CLICK RECORD!!!!!!!!!!!!!!!!! For HTS Mode, you can select all desired wells and click Run Wells, or you can run each manually with the Acquisition Dashboard using Acquire Data and Record Data. Data files will automatically be saved. An icon with a floppy disk will appear when the sample has been run and saved. or 19

20 9. Exporting and Transferring Recorded Data After you finish recording your last sample, export your Experiment and FCS files. Right click on the name of your experiment and in the menu select Export, then FCS Files. Make sure you are exporting FCS 3.0 files, then click OK. In the window that appears, click the Browse button, then select D:\BDExport\FCS as the destination folder, Browse and find (or create) a folder for your lab group. Then click Export to save your files there. Files can now be transferred off the computer via PASS Space, DropBox, etc. The D drive, where exported files are stored, can be accessed using a shortcut on the Desktop. 20

21 10. Cleaning after HTS Mode After finishing your data acquisition, prepare an empty plate (stored near sink) for cleaning: Fill wells A1-A4 with FACS Clean/10% bleach Fill wells B1-B4 with Distilled Water Fill a small flask with Distilled Water Disconnect the clear/white sheath line from the HTS Sheath port by pressing the metal button facing the main Fortessa instrument. Click a short HTS purging tube (stored near the sink) into the sheath port. Sometimes you need to push on the button to get it to snap in. Place the opposite end of the short tube into the flask of Distilled Water, ensuring that the tube is deeply submerged. Load the plate onto the HTS. Put the cytometer in Run. In Diva, click HTS Clean. Click OK. Then, when Diva says to install the cleaning plate, click OK and the plate will automatically run. It will take about 10 minutes. When the plate is finished being run, put the cytometer in Standby. Return the tubing to its original state: Disconnect the short HTS purging tube from the HTS sheath port, and reinstall the sheath line into the port. Rinse out the plate with distilled water and return to storage spot near sink. Empty the flask, and store the flask and HTS purging tube near the sink. 21

22 11. Cleaning after Tube Mode After finishing your data acquisition, prepare 2 tubes for cleaning Tube 1 (25% Full): FACS Clean/10% bleach, Tube 2 (25% Full): Distilled Water. Place the cytometer in Standby and HIGH. Run Tube 1 (FACS Clean) on High with the SIT arm on the side for 1 minute. Run Tube 2 (Distilled Water) on High with the SIT arm on the side for 1 minute. Next, return the cytometer to HTS Mode. Place the cytometer in Standby. Unscrew the conical nut at the top of the DCM sleeve. Carefully remove the DCM sleeve by gently sliding it straight down off of the SIT. Place the DCM sleeve in the small tube attached to the side of the cytometer, and get the SIT protector out of this small tube. Slide the SIT protector up over the SIT until it reaches a hard stop. Screw it onto the SIT. Attach the HTS sample coupler thumbscrew to the SIT. First, ensure that the white section is securely screwed in to the brown section. Then, screw the brown thumbscrew into the remainder of the brown section. Then, unscrew the brown thumbscrew ½ turn so that it is loosened. In this loosened state, slide the sample coupler onto the SIT until it reaches a soft stop THEN, push a bit more until it reaches a hard stop. Hold the lower part of the brown section with one hand while you tighten the brown thumbscrew with the other hand. Switch the acquisition control on the side of the cytometer to HTS/Plate Mode: Turn ON the power to the HTS using the switch on the back of the HTS. Ensure that the cytometer is in Standby 22

23 12. Batch Analysis Now you may choose to perform a batch analysis. It will give you the option to print multiple worksheets or save them as a PDF file. It will also allow you to export the data from the Statistics View to a CSV file that can be read in Excel. To start a batch analysis you need to right click on your Experiment name (or Specimen, or several selected tubes) and select Batch Analysis. Then select the actions you want the software to perform. Also, it is important to specify the destination where your PDF and CSV files are going to be saved. Click the Browse button and select the same folder as the experiment on the desktop in both cases. Copy your folders to a storage device and proceed with the cleaning procedure. 23

24 13. Logging Out from the Software To log out from the software, select Log Out from the File menu. 14. Shut-Down When all finished with running samples, cleaning, and analysis, shut down the system unless a staff member is in the room or the next person signed up for the machine is in the room. To shut off: 5. Turn off the computer. 6. Turn off the cytometer with the green button. 7. Turn off the FACS Flow Supply Cart on the floor with the green button. If a staff member is present or the next user is in the room, place the cytometer in Standby. 24