Mar 20, 2025
SOI - BD FACSAria Fusion Cell Sorter - Sorting Operation
This protocol is a draft, published without a DOI.
- 1European Molecular Biology Laboratory (EMBL)
Protocol Citation: Daniel Gimenes 2025. SOI - BD FACSAria Fusion Cell Sorter - Sorting Operation. protocols.io https://protocols.io/view/soi-bd-facsaria-fusion-cell-sorter-sorting-operati-d52d88a6
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: March 20, 2025
Last Modified: March 20, 2025
Protocol Integer ID: 124709
Keywords: Cell sorting, Flow Cytometry, Core Facility, FACSAria Fusion
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Abstract
This Standard Operation Instruction is intended to guide the facility users to operate and perform cell 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 sort particles in the cell sorter.
Perform the steps subsequently unless otherwise stated.
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.
Creating and editing a Sort Layout
Creating and editing a Sort Layout
On the “Browser” window, click on the sort layout button:
On the sort layout window, make sure you have the correct “Device” selected that matches your
collection tubes/plates:
- 2 Tube: 15 mL Tubes Collection device (max. 2 populations at a time)
- 4 Tube: 5 mL tubes and 1,5 mL tubes. (max. 4 populations at a time)
- 6 well plate (Custom)
- 24 well plate (Custom)
- 96 well plate (Custom)
- 384 well plate (Custom)
On “Precision” select the desired mask for your sorting.
- If going for Yield, select Yield.
- If going for Purity, select 4-way-purity (even if you are doing a 2-way sorting!)
- If you are doing single cell sorting, select Single Cell.
On “Target Events”, leave on continuous to sort indefinitely or type the desired amount of collected events.
Note
Important: The Single Cell option is a sorting mask and not a number, so if you are doing single cell sorting, add “1” to this field to have only 1 particle sorted per well.
On “Save Sort Reports”, select the option “Save all’.
After gating your populations of interest, assign the tubes/wells you want them to be sorted into.
Note
Important: pay attention if the “Target Events” value matches the value on the assign tubes/wells.
Put your tube(s) or plate into the collection device under the sort block.
Start the acquisition, press “Sort” in the sorting layout window. Be sure to click “OK” to move the waste drawer to SORT position!
Note
Important: The “Sort” option doesn’t automatically save your data, so if not done yet, be sure to also press “Record” on the “Acquisition Dashboard”!
Monitoring the stream
Monitoring the stream
Regularly check the values for Gap and Drop 1.
If the actual Drop 1 value is ± 11 compared to the target value, stop the sorting and re-define the drop delay (see how to set the stream and determine the drop delay value in the Fusion Start up protocol).
Protocol
NAME
SOI - BDAria Fusion Cell Sorter - Instrument Startup
CREATED BY
Daniel Gimenes
If the sweet spot is not maintaining the actual Gap value in range (±2) compared to the target value or if the Drop 1 is shifting between drops, stop the sorting, find a new stable amplitude for the stream and redefine the drop delay (see how to set the stream and determine the drop delay value in the Fusion Start up Protocol - see also the reference picture and values for your correspondent nozzle in the Fusion Protocols folder).
Monitoring the threshold rate
Monitoring the threshold rate
Frequently check and adjust your flow rate according to the sorting efficiency. (Try to keep this value over 80% at all times unless doing high throughput sorting – if not possible, stick to the highest obtained value).
Typical threshold values (as a reference, adjust as necessary):
- 130 μm: 3000 events/s
- 100 μm: 5000 events/s
- 85 μm: 9000 events/s
- 70 μm: 15000 events/s
Avoid going way over these recommended threshold rates. This can compromise efficiency and clog the
nozzle!
Avoid going over a flow rate of 5.0 unless doing high throughput sorting. If necessary, opt instead to concentrate your sample.
Moving to the next tube
Moving to the next tube
On the “Acquisition Dashboard”, press “Unload”. This will stop both the sorting and acquisition.
Remove the tube from the sample loading port.
Write it down the number of sorted droplets in the worksheet.
Save a PDF in case you are going to change the position of the gates in the next sample.
Remove the collection tubes and either vortex or invert them to mix with your collection medium.
Load a tube of dH2O for at least 30 seconds at a Flow Rate of 11.0.
Perform a Sample Line Fluidics Back flush for at least 30 seconds.
Add your new collection tubes (be sure there are no bubbles on top) to the sorter collection
device.
Vortex and load your sample in the loading port.
Adjust the gates and the sorting layout if necessary, then press “Sort” and “OK”.
Press “Record” on the “Acquisition Dashboard.
Loading a new tube of the same sample
Loading a new tube of the same sample
Click on “Unload”. This will automatically stop both the acquisition and sorting (it might take a couple of seconds until the software responds).
Remove your sample tube from the sample loading port.
Put your next sample tube in Sample Loading Port and press “Load”.
Changing to a new collection tube
Changing to a new collection tube
Click on “Stop Acquisition”.
Take your collection tubes and invert them a couple of times to mix the sorted drops with the medium (getting rid of the "machiatto" effect).
Add the new collection tubes into the collection device.
Note
Important: Notice if there are any bubbles on top of the collection medium!
Click on “Acquire” in the Acquisition Dashboard.
On the Sort Layout Window, click either on “Sort” (counting will re-start from 0) or “Resume” (counting will continue from where it left off).