Mar 25, 2025
SOI - BD FACSAria Cell Sorter - Troubleshooting guide
This protocol is a draft, published without a DOI.
- 1European Molecular Biology Laboratory (EMBL)
Protocol Citation: Daniel Gimenes 2025. SOI - BD FACSAria Cell Sorter - Troubleshooting guide. protocols.io https://protocols.io/view/soi-bd-facsaria-cell-sorter-troubleshooting-guide-d52688he
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 25, 2025
Protocol Integer ID: 124734
Keywords: Flow Cytometry, Cell Sorting, Core Facility, Troubleshooting, FACSAria Fusion
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This Standard Operation Instruction is intended to guide the facility users to troubleshoot problems in 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 an error arises or a problem arise.
Perform the steps subsequently unless otherwise stated.
Software Troubleshooting
Software Troubleshooting
Problem: Instrument and software do not connect.
Description: After starting the software, PC and instrument should connect after a few minutes. Sometimes the connection fails and one cannot use any acquisition function on the instrument. Most of the time this error appears if one doesn’t follow the correct order of PC → Instrument → Software during startup.
Solution:
- After 3 min if still no connection, turn the instrument and PC off.
- Wait for a min, switch on PC, log into Windows.
- Switch on the instrument and wait for the tube agitation motor to spun two times.
- Open the software.
Problem: DIVA software is always on top.
Description: When trying to open CST or other windows in the computer, DIVA stays always on top and has to be minimised in order for one to see the other open windows.
Solution: Go to “File” → “Print” (or Ctrl+P). Close the Print window.
Problem: Tube is stuck in the sample injection chamber.
Description: Sometimes after loading a tube, an error message appears and the Unload function is not available.
Solution:
- Press the red emergency button located in front of the instrument (see the Nozzle clog troubleshoot session). The stream will stop and the tube will be unloaded.
- Restart the stream (check the Startup protocol for guidance on how to set the stream for sorting).
Protocol
NAME
SOI - BDAria Fusion Cell Sorter - Instrument Startup
CREATED BY
Daniel Gimenes
Acquisition Troubleshooting
Acquisition Troubleshooting
Problem: No events
Description: The tube with the sample is loaded, but no events are displayed on the dot plots.
Solution:
- The laser interlock is open → If the laser interlock is open, the lasers are automatically shut down → close the interlock.
- The sample line is clogged → First activate the sample line back flush (Cytometer → Cleaning Modes → Sample Line Back flush). Let it run for a minute. Load a tube and check if the instrument can now detect events. If not, proceed to the steps below.
Silicone tubing massage → Unload the tube and activate the Sample Line Back flush.
Once the back flush is activated, the pinch valve will go down. Pull out the sample line out of the pinch valve and bubble detector (see below illustrations). Stop the back flush.
In the sample injection chamber, check if the sample line is dripping. If yes, the sample line is not clogged. If no dripping is observed, the sample line is clogged.
Massage the silicone tubing until the sample line is again dripping. If the massage doesn’t solve the issue, the facility staff will have to replace the sample line.
Click again on “Start” to restart the back flush, then push the silicone tubing back in the pinch valve and bubble detector. Stop the back flush. If the silicone tubing is in the correct position, no dripping should be observed.
Sorting Troubleshooting
Sorting Troubleshooting
Problem: Low efficiency.
Description: The sorter efficiency is the relationship between the sorted events divided by the number of sorted events + aborted events. A low efficiency means that potential target events are being wasted.
Solution: Try the following steps:
The threshold rate is too high → lower the flow rate. If already on 1.0, unload the tube and dilute your sample.
Too many aggregates in your sample can cause abrupt shifts in the threshold rate → filter your sample again and unload the tube to vortex it from time to time if doing a long sorting.
The sort mask precision is wrong: → Select the appropriate precision mode (e.g., Single Cells to 4-way Purity when doing bulk sorting).
The nozzle is too small → Try using a larger nozzle (if possible).
Problem: Nozzle clog.
Description: Debris, cell aggregates and air can block the nozzle orifice and cause an interruption of
the stream.
Solution: Try the following steps:
If the stream didn’t automatic stopped, switch it off by pressing the green check mark button in the
Stream Window (if the stream stopped automatically, proceed to go to step #6.3 ).
If the button is not available, proceed by pressing the Emergency Stop Button located in front of the instrument.
Note
Rotate the emergency button clockwise and confirm all the warning dialog that pops up.
Remove the nozzle from the nozzle holder, turn the nozzle upside down (red o-ring facing down) and using the 5 mL syringe with the yellow tip, rinse the nozzle orifice with dH2O.
Dry the nozzle with paper towel or compressed air (be careful with the o-ring!)
Insert the nozzle back and close the nozzle lock.
Restart the stream by pressing the Red Cross in the Stream Window.
Check if the stream position is right over the waste drawer (if not, repeat the procedure from 3-6).
The stream might need adjustment on the Amplitude to return to the same Drop1 value. If after adjusting the Amplitude, the original Drop 1 value changed, rerun the Drop Delay (for instructions, check the Startup SOI).
If you suspect that the cause of the clog was your sample, re-filter it to prevent new clogging.