Mar 17, 2025
SOI - BDAria Fusion Cell Sorter - Instrument Startup
This protocol is a draft, published without a DOI.
- 1European Molecular Biology Laboratory (EMBL)
Protocol Citation: Daniel Gimenes 2025. SOI - BDAria Fusion Cell Sorter - Instrument Startup. protocols.io https://protocols.io/view/soi-bdaria-fusion-cell-sorter-instrument-startup-dtbw6ipe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 04, 2024
Last Modified: March 17, 2025
Protocol Integer ID: 113750
Keywords: Flow Cytometry, FACSAria Fusion, Cell Sorting, Core Facility
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Abstract
This Standard Operation Instruction is intended to guide the facility users to perform a fluidics start-up on a BD FACSAria Fusion™ instrument
Guidelines
This protocol should be done by:
- The faciity staff
- Trained users of the FCCF.
This protocol should be performed:
- Everyday (assuming a Fluidics shutdown was done on the previous day).
Perform the steps subsequently unless otherwise stated.
Materials
- BD FACSDiva™ CS&T Research BeadsBecton Dickinson (BD)Catalog #655051
- BD FACS™ Accudrop BeadsBecton Dickinson (BD)Catalog #345249
- FACSCleanBecton Dickinson (BD)Catalog #340345
- BD FACSFlow™ Sheath FluidBecton Dickinson (BD)Catalog #342003
- FACSRinse (discontinued by the manufacturer
- Distilled H2O
Safety warnings
Handle FACSClean carefully. It should't enter in contact with the skin and eyes (wear gloves, lab coat and glasses) and it ruins colorful clothes.
Before start
Be sure to wear gloves and lab coat at all times!
General rule: spray 70% ethanol in all connectors before plugging any tubing!
Preparing the workspace
Preparing the workspace
Turn on the PC
Login in the Admin account (Password: attached to the PC screen).
Manually open the biosafety cabinet window and make sure that the ventilation is off). Wipe the surface in front of the instrument with 70% ethanol
Safety information
Spray the 70% ethanol first on a paper tissue and not directly on the safety cabinet to not create potential harmful aerosols.
Block the keyboard by pressing the Fn + Clean on/off buttons. Wipe the keyboard and mouse with 70%
ethanol
Turn on the instrument by pressing the green button on the lower right side.
Turn on the safety cabinet by pressing the “ventilation” button on the upper right panel and
adjust the position of the window until it reaches the arrows.
Turn on the safety cabinet light by pressing the “light bulb” button.
Servicing the tanks
Servicing the tanks
Open the lower right metal door in the instrument. Pull the fluidics cart by pressing the small
yellow lever on the right upper part of the cart.
Unplug the connections and remove the waste tank from the fluidics. Discard the contents
of the waste tank on the sink. Return the waste tank to the fluidics cart and plug
back the connections.
Check the levels of the sheath fluid tank in the software. If refilling is necessary, first remove any residual pressure. Spray 70% ethanol on the lid of the sheath tank before opening it. Refill it with sheath fluid until it reaches the top marking inside the tank. Close the lid.
Remove the fluidics tubing (blue tubing labeled with a red tag that reads ‘Fluid’) that comes after
the ethanol filter (labeled with red tag that reads ‘Fluid Shutdown’) and plug into the sheath filter (labeled with a red tag that reads ‘Fluid Start-up’). Remember to always spray 70% ethanol when exchanging connections.
Note
Check the instrument folder for pictures if in doubt!
Remove the air connection (labeled with a green tag that reads ‘Air’) from the metal ethanol tank (labeled green with a tag that reads ‘Air Shutdown’ and plug into the sheath tank (labeled with a green tab that reads ‘Air Start-up’).
Note
Check the instrument folder for pictures if in doubt!
Push back the fluidics cart and close the metal door.
Fluidics Startup
Fluidics Startup
7m
7m
Start “BD FACSDiva Software”.
Login into the “Administrator” account (no password required).
Wait for the software connect to the instrument - on the right bottom corner it will change from Connecting… (yellow dot) to Connected (green dot).
When the CST-Mismatch window dialog appears, select: “Use CST Settings”.
Go to “Cytometer” → “Fluidics Startup”. Follow the instructions on the screen:
Confirm that the fluidics tubing is connected to the sheath fluid filter and that the air tubing
is connected to the sheath tank and press “Done”.
Confirm that the closed-looped nozzle is correctly installed into the flow cell (the red o-ring
should be facing up!) and press “Done”.
The Fluidics Startup will now begin and should take approximately 00:07:00 minutes.
7m
While the Fluidics Startup is running:
Prepare fresh cleaning tubes: Rinse, FACSClean and dH2O (from the 50 mL syringe
with a filter!)
Rinse the nozzle orifice with dH2O: Pour dH2O (from the 50 mL syringe with a filter) into a 50 mL Falcon Tube. Grab the 5 mL syringe with the yellow tip and fill it with the tube water. Take the nozzle and turn it upside down (with the red o-ring facing down!). Place the tip over the nozzle orifice and gently rinse it until it’s possible to see liquid coming through in a form of a stream.
When the Fluidics Startup reaches around 70%, check if the sample line is dripping from the
inside (to detect a potential clogging).
Once the Fluidics Startup is done, open the nozzle lock (by turning it anti-clockwise until 8:00 position) and remove the closed-looped nozzle. Confirm on the software by pressing “Done”.
Install the dry nozzle with the red o-ring facing up. Close the nozzle lock (by turning it clockwise until the 12:00 position) and confirm on the software by pressing “OK”.
Changing Configurations
Changing Configurations
Check if you currently have the right configuration for the nozzle you are going to use (look at the top bar of the software).
In case you need to change the configuration, go to “Cytometer” → “View Configurations”.
Select the desired configuration between the available on
Press first “Set Configuration”.
And then "OK".
Check if the desired configuration was loaded.
Exit the CS&T module by clicking on the "File → Exit"
Setting up the stream
Setting up the stream
On the Stream Window start the stream by pressing the “red cross”.
Wait until the stream forms and it's possible to see the drops on the image. The stream could take some minutes to completely form.
Open the door of the sort chamber. Check if the stream is hitting the centre of the aspirator. If not, take the black/yellow screw driver and loose the two screws on the both sides of the sort block chamber.
Grab below and gently move the sort chamber to the sides until then stream hits the centre of the aspirator. Using the screw driver, re-tight (lightly!) the sort chamber screws.
Close the sort chamber and the upper cover of the instrument.
Go to “Cytometer”, “Cleaning Modes”, “Sample Line Backflush”.
On the Sample Line Backflush window, press “Start”. You should see sheath fluid flushing from both
the inside and the outside of the sample line. If the sample line is not dripping from the inside, perform the “tubing massage” (Troubleshooting SOP). Stop the Sample Line Backflush by pressing “Cancel”.
Cleaning the sample line
Cleaning the sample line
Double click the Accudrop Drop Delay Worksheet and select Tube_001.
Load and run sequentially Rinse, Clean and dH2O solutions for 5 minutes each at a flow rate of
at least 4.0.
Running the CST Beads
Running the CST Beads
Go to “Cytometer” → “CST”. Diva will disconnect and the CST module window will open.
Check if the ND filter 1.5 is installed in front of the FSC detector (if the 2.0 is installed, swap it for the 1.5).
Make sure the option "Check Performance" is selected, and press “Run”.
Put the tube containing the CST beads into the loading port and confirm by pressing “OK”. The
program will now automatically check and adjust the settings.
If the performance check fails or gives a warning, check the Troubleshooting protocol.
Exit the CS&T module by clicking on the "File → Exit". Wait a few seconds for Diva to reconnect.
Adjusting the Stream
Adjusting the Stream
Look the overall shape of the stream. If the drops are presenting weird shapes, or if the satellite droplets are not joining the large droplets, stop the stream and clean the nozzle (Troubleshooting protocol). Restart the stream. Otherwise proceed to the next step.
Source: BD FACSAria Manual
Adjust the amplitude slider until the drop break off is happening about four to five (for the 70 and
85 μm) or three to four (for the 100 and 130 μm) drops from the top of the break off window.
Fine tune the amplitude until the Gap “Actual Value” matches the “Target Value”.
Type the value of the “Actual Drop 1 Value” to the “Target Drop 1 Value (white box).
Once the Drop 1 value is stable, activate the Sweet Spot (be careful to not accidentally click on the
Stream button!).
Determining the Drop Delay
Determining the Drop Delay
Adjust the Accudrop diode laser by turning the micrometer dial clock or anti-clockwise
until the brightest and biggest spot is seen in the side stream window.
Open the Accudrops tube Sort Layout window from the Drop Delay Experiment.
Make sure the Device is set on “2 Tube”, the Precision is set on “Fine Tune” and the Target Events
is set on “Continuous”. The sorted population should be NOT(P2) on the Left.
Put the tube containing the Accudrop beads into the loading port and load it. Start with 1.0
and adjust the flow rate according to the nozzle you are using in the Acquisition Dashboard.
On the side stream window, activate the “Optical Filter”.
The displayed layout will change and it will be possible to see two squares. You should see 100% on the right square (if not, consider increasing the concentration of the beads, the flow rate, or re-adjusting the diode laser focus).
Turn on the voltage of the deflection plates.
Press “Sort” in Sort Layout Window. When prompted, select cancel.
Move the left slider until the deflected stream is inside the centre of the left square
Click on the “Auto Delay” button in the Side Stream Window.
On the Auto Drop Delay window, click on “Start Run”.
The instrument will calculate the best Drop Delay Point and apply it to the experiments. The
percentage of deflected beads should be higher than 95% on the left square.
Unload the tube containing the Accudrops.
Setting up the sort deflection
Setting up the sort deflection
Open the "Sort Device Control" (you can find the shortcut in the desktop).
Select the desired sort device (ACDU for plates, 4 tubes for FACS tubes, Two tubes – 15 mL Falcon
tubes, 1,5 mL tubes – Eppis). Don’t change any other setting in this window!
Turn on the voltage of the deflection plates on the Side Stream Window.
Click on the “Test Sort” button.
Move the sliders until the deflected streams are inside the correspondent parallel bars (if one or more side streams are not visible/bright enough, consider re-adjusting the diode laser focus).
Deactivate test sort. Position the test collection tubes into the correspondent collection device.
Close the “Waste Drawer”.
Double click “Test Sort”.
Check the top of the tubes to check a droplet in the centre. If not, readjust the deflection sliders
and repeat the process.
Deactivate the “Voltage” and reopen the “Waste Drawer”.