Aug 25, 2025

Public workspaceSOI - BD FACSAria Fusion Cell Sorter - Plate Sorting

This protocol is a draft, published without a DOI.
SOI - BD FACSAria Fusion Cell Sorter - Plate Sorting
  • Daniel Gimenes1
  • 1European Molecular Biology Laboratory (EMBL)
Daniel Gimenes
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Protocol Citation: Daniel Gimenes 2025. SOI - BD FACSAria Fusion Cell Sorter - Plate Sorting. protocols.io https://protocols.io/view/soi-bd-facsaria-fusion-cell-sorter-plate-sorting-d69y9h7w
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: April 02, 2025
Last Modified: August 25, 2025
Protocol Integer ID: 125976
Keywords: Flow cytometry, FACSAria Fusion, Cell sorting, Core Facility, Plate sorting, bd facsaria fusion cell sorter, bd facsaria fusion, fusion, plate, standard operation instruction
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Abstract
This Standard Operation Instruction is intended to guide the facility users to set-up and perform plate sorting on a BD FACSAria Fusion™ instrument
Guidelines
This protocol should be done by:

  • The facility staff.
  • Trained users of the FCCF.

This protocol should be performed:
  • Every time one wants to perform plate sorting in the cell sorter.

Perform the steps subsequently unless otherwise stated.
Materials
  • Splash shield
  • PCR plate holder (if sorting on PCR plates or strips)
  • ReagentBD FACS™ Accudrop BeadsBecton Dickinson (BD)Catalog #345249
  • 96 well plate covered with plastic foil

Protocol materials
ReagentBD FACS™ Accudrop BeadsBecton Dickinson (BD)Catalog #345249
Troubleshooting
Before start
  • Perform a Fluidics Startup (SOI - BDFACSAria Fusion Cell Sorter - Instrument Startup).
  • Make sure the stream is adjusted and stable.
  • Create an experiment, adjust the PMT gains and run compensation (if necessary).
  • Create a gating strategy to reach target population.
Installing the splash shield
Make sure the sort block chamber is closed. Remove the tube collection device, if installed.





Insert the splash shield into the slotted fittings below the sort block chamber and push it all the way in.




Note
Please note that the magnet on the splash shield must be facing the upper right position




Open the Drop Delay experiment (FCCF → Set-up → Drop Delay). Expand "Global Worksheets" and "PLEASE DON'T EDIT THESE SORT LAYOUTS!!!". Double click on the correspondent sort layout to open it according to your plate option:


The available options are the following:
  • 6 wells
  • 12 wells
  • 24 wells
  • 96 wells custom (100 µM) - Partial
  • 96 wells custom (130 µM) - Partial
  • 384 wells custom - Partial
To bring the ACDU tray (Automated Cell Deposition Unit) to the front of the instrument, click the Access Stage button in the sort layout.



Pull the lever to the left and place over the ACDU tray a test plate related to the plates you are going to sort. The A1 position should be toward the front of the stage on the left side.




Note
The sorting order is A1 to the back of the plate and then from left to right and back to the front, in a zig zag motion. This means that a full 96 wells plate will have A1 as first well and H12 as last.

Adjusting the side stream deflection
Open the Sort Device Control.



On "Device, select "ACDU". Do NOT change any other settings! Close the software.



Collapse the left, right and far right side streams.




On the side stream window, activate Voltage. The icon will become red to indicate that the deflection plates are being charged.



Activate Test Sort.





Adjust the far left side stream until it is in the centre of the the two left parallel bars on the side stream camera. Finally, switch off Voltage.




Adjusting the plate position
Go to Sort → Custom Devices.



Find the custom option for the device you want to sort into.



Click on Go to Home. The tray is going to move to the home position.




Activate Voltage in the side stream window.



Double click the Test Sort button in the Device Setup window to deposit a drop over the plate at the home location.

Need a screenshot with the test sort activated
Check the plate to see if the drop was deposited in the centre of the well.


If necessary, adjust the coordinates for the home location. Click on the arrows to move the tray to the desired position.



Note
Please note that when you are clicking on the arrows, what it is moving is the tray with the plate, and not the stream! This means that for instance - if a drop is too much to the right of the well - the tray has to be moved to the right so that the drop is deposited more to the left.

The small arrows move the tray by one step and the large arrows by 5 steps.
Repeat Go to through Go to until the deposited drop is in the centre of the well.

Click on Set Home.



Note
Be careful to NOT click on Set Farthest!!!

Click on Go Farthest. The plate will move to the farthest well from A1.



Repeat Go to through Go to until the deposited drop is in the centre of the well.

Click on Set Farthest.



Switch off the voltage.



Testing the drop deflection
In the Drop Delay Experiment, double click to open the sort layout for the device you just have set.



Note
If testing a 96 or 384 wells plate, one can test first the "Partial" layout to speed up the time of sorting for checking.

Put the tube with ReagentBD FACS™ Accudrop BeadsBecton Dickinson (BD)Catalog #345249 into the loading port. Load the tube.



Click on "Sort".


A Confirm dialog will follow. Press OK to confirm.



Check if the drops are in the centre of the wells. If not, repeat Go to to Go to until the drops are properly placed.