University of Utah Flow Cytometry Core Facility SOP 14.0

University of Utah Flow Cytometry Core Facility SOP 14.0 BD Fortessa User Guide 9-22-16

BD Fortessa OPERATION GUIDELINES

INSTRUMENT START-UP:

1. Turn on cytometer main power (right side of instrument) and computer. 2. Log on to Windows using the password your UID and password if you are and HCI member. If not, use username: fortessauser Password: Welcome! 3. Start (double click) the FACSDiva software icon. Ideally the instrument should be “warmed up” for 10min. 4. Check fluids and fill/empty as needed. 5. Log on to the Diva software (using your username and password). The software should open with windows for “Browser”, “Inspector”, “Cytometer” and “Acquisition Dashboard” on the left screen and a window for “Worksheet” on the right screen. a. If any of these windows is not open. Select “View→choose whatever window you need”#1 6. Wait for the software to connect to the cytometer (See bottom of “Cytometer” window #2). 7. The instrument is now ready to run samples.

Fluidics Panel

Run=Pressurizes tube, sample will immediately be run through instrument. Does not make a difference if “Acquire” was selected in software

Standby=Does NOT pressurize tube and nothing is running through the instrument

Prime=To be used when instrument is clogged or apparent bubbles in the tubing. Place sample arm to the side and press “Prime”. Process takes about 15 seconds and will default to “Standby” when completed

Low~30ul/min Med~60ul/min High~120ul/min

PREPARING TO RUN SAMPLES:

If creating experiment from scratch: 1. In the “Browser” window #4, create a new experiment by clicking the icon. #5 2. Highlight the Experiment and rename 3. Select the experiment-level settings #6 and change, add or delete parameters as needed in the Inspector Window #7. Also choose Lin/Log and Area, Height, Width (A,H, W) for each parameter 4. If you have saved instrument settings load these by right clicking the experiment level settings #6, choosing “Apply from Catalogue”, selecting a settings file and clicking “Apply”. University of Utah Flow Cytometry Core Facility SOP 14.0 BD Fortessa User Guide 9-22-16

5. Select Experiments > Compensation Setup > Create Compensation Controls. #8 6. Select unstained tube from “Compensation” Specimen. #9 7. Put unstained tube on instrument and select Acquire #10 8. Adjust all voltages in Cytometer Window #11 9. Adjust P1 gate #12 to include cells if interest, then, right click in gate and “apply to all compensation controls” 10. Adjust stopping Gate and Event number #13 11. Select “Record”#14 12. Select Next Tube #15 13. Acquire and record single color control tubes. Upon completion be sure each positive peak in each histogram is included in the gate for every tube. 14. Select “Experiment”#8 > “Compensation Setup” > “Calculate Compensation”. Name and save your compensation settings by clicking on “Link and Save” to load compensation. 15. Double click on the worksheet icon (top left) to convert the Normal Worksheet (for compensation) back into a Global Worksheet (for data acquisition). #16 16. Create plots and gates needed for analysis #17

Importing Templates 1. File→Import→Experiment (D:BDEXPORT/Experiment/PI Folder) 2. Rename experiment

RUNNING SAMPLES:

1. Add a new Specimen #19 2. Select your first tube by double clicking on the tube pointer (be sure it changes to a green highlighted arrow). #20 3. Enter identifying sample information in either of two ways: a. Stepwise by adding tubes one at a time and then changing the tube name b. For an entire batch of tubes by entering all labels and information in “Experiment” > “Experiment Layout”. #8 4. Load your first sample on the SIT and click on “Acquire Data” #10. As soon as flow stabilizes (≈5 sec), and if everything is adjusted to your satisfaction, adjust stop criteria and then click “Record Data” #14. The sample will run until the stop point is reached. 5. Swing sample arm to the side and remove your sample tube. Leave sample arm to the side in between samples in order to backflush. If entering tubes manually, click on “Next Tube”#15, enter new sample information repeat steps 3-4. 6. Collect data for all tubes 7. Export FCS Files. Highlight Experiment→ File→ Export→ FCS Files (D:BDExport/FCS/PI Folder) 8. Save templates

ANALYZING DATA FILES: University of Utah Flow Cytometry Core Facility SOP 14.0 BD Fortessa User Guide 9-22-16

1. Right click on the Specimen that holds the tubes you want to analyze. 2. Select “Batch analysis” 3. You will have the option to manually go through the files and adjust gates during batch analysis or automatically go through them using the gates that are currently displayed in the worksheet. 4. Choose either manual or Auto. If you choose Auto, select view time to 0. 5. Select PDF and or Stats for export. 6. Change destination folder and file name by selecting browse where appropriate. 7. Select “Run”

Saving Templates 1. Right Click Experiment, select Duplicate without data. 2. Save this new experiment with a new name (example GFP TEMPLATE) 3. Right Click on experiment, Export→Experiment. (D:BDEXPORT/Experiment/PI Folder) 4. Delete Experiment from Browser after export