Using the BD LSRFortessa
Penn State
Microscopy & Cytometry Facility
Flow Cytometry lab
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
6.10. Setting Up the Number of Events to Record
6.11. Setting Up Voltages Based on an Unstained Control
6.12. Adjusting Area Scaling for FSC
6.13. Adjusting Area Scaling for Each Laser
6.14. Checking the Brightest Tubes
6.15. Saving Application Settings
6.16. 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
1. Start-Up
1.
2.
3.
4.
Turn on the FACS Flow Supply Cart on the floor with the green button.
Turn on the cytometer with the green button.
Turn on the computer.
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
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
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
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.
4.2. 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.
4.3. 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
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.
5.2. 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.
5.3. 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
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
6.2. Selecting H as a Parameter
Check “H” for all parameters except SSC.
6.3. 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.
6.4. 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
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 “P1”.
6.6.
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
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.
6.8.
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
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
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
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
Scaling” for optimal
settings
Increase “FSC Area
Scaling” for optimal
settings
14
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
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.
6.15. 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
6.16. 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
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.
7.2. 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
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.
7.3. 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
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
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
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
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
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
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