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  • FACSAriaIII system
  • Biosafety Safety Cabinet
  • Sonicator
  • PPE including gloves, long sleeve gowns, P2 respirator, safety glasses, enclosed shoes
  • Bleach (12.5%) (Diluted 1 in 10 final)
  • Decon 90 (concentrated surface decontaminant) (Diluted 1 in 20 final)
  • Ethanol (70% w/w)
  • Trigene (Diluted 1:50100) or F10SC (Diluted 1:250) if Trigene isn’t available.

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  1. Turn on the CAS BSC hood, chiller, high-pressure gas and aerosol management system (set at 20% - for operational velocity evacuation).
  2. Turn on the main switch to the ARIAIII located on the left of the instrument. 
  3. Log into Windows using provided credentials. 
  4. Log into the PPMS account with provided credentials. 
  5. BD FACSDiva should launch automatically as should Google Chrome (contains the sorting log).
  6. Log in to BD FACSDiva, the software will detect and connect to the instrument. If it does not do it automatically, click Instrument in the main menu and connect the instrument manually.
  7. If a CST mismatch dialog appears always click ‘Use CST Settings’
  8. Open the BCS cabinet.
  9. Open the front cover of the AriaIII and prob prop it open.
  10. Determine if the correct nozzle is in place in the flow cell.
    1. If the cleaning nozzle is in place perform a fluidics startup (as this indicates the fluidics was shutdown).
    2. If the incorrect nozzle is in place in the flow cell, sonicate the appropriate nozzle in DI water for 5 minutes in a 5mL FACS tube. Ensure sonicator has sufficient liquid in it to conduct cleaning power to the nozzle.
      1. If needed - unlock and remove the previous nozzle or waste nozzle from the flow cell. Place the waste nozzle into an empty slot on the nozzle storage block.
      2. Use a Kimwipe to remove any residue liquid on the sonicated nozzle to be installed if necessary.
      3. Clean any salt residues around the nozzle assembly area using ethanol. Wipe and dry clean. 
      4. Insert sonicated nozzle with the O-ring facing upwards. Lock the nozzle using the nozzle clip.
      5. Since the nozzle was changed, also change nozzle size settings in the software by setting the correct cytometer configuration - >Cytometer>Configurations>Select correct nozzle>Apply. 
      6. Open the sort block door, click the stream start/stop button to turn on the stream.
      7. Check the stream video feed is not impeded by any liquid/debris on the lens. If this is the case it can be cleaned with a lens cleaning tip.
      8. Check the stream angle. If the stream flows away from the centre of the waste catch, loosen the screws on both sides of the collection assembly and rotate the sort block to adjust.
      9. Close the sort block and thumb screw it shut.
    3. If the correct nozzle is in place in the instrument, ensure the correct instrument configuration is selected and applied in the software by looking at the window title bar. 
  11. The ARIAIII flow cell cover should be open.
  12. Open the sort chamber (we need to view whether the stream is aligned or not. 
  13. Close the front cover of the BSC.
  14. Be ready to change the waste catch position and then start the stream.Change the waste catch position if necessary. catch position and then start the stream.
  15. Check the stream angle. If the stream flows away from the centre of the waste catch, loosen the screws on both sides of the collection assembly and rotate the sort block to adjust.
  16. Close the sort block and thumb screw it shut.
  17. Stop the stream. This starts still results in air into the sheath tank which is used to minimise contamination of the sheath tank when refilling. 
  18. Unscrew the sheath tank lid completely.
  19. Remove the air pressurising line.
  20. Relieve all pressure from the tank by venting using the venting valve, and breaking the air-pressure seal by pushing down. Ensure you keep fingers clear of the lid and remove it. It can be stored temporarily on the fluidics cart (keeping the inner face not touching anything).
  21. Connect the air pressurising line promptly to minimise contaminants falling into the tank. 
  22. Fill sheath tank to the weld mark (~10cm from top of tank).
  23. Reseal sheath tank - only holding the screw mechanism to keep fingers clear of the lid. 
  24. Empty waste container slowly into the sink under running water. Put 100ml of bleach into the waste tank.
  25. Reconnect waste container.
  26. Check the filter attached to the sheath and at the front of the fluidic cart for air bubbles. If needed, ethanol spray and then gently bleed out any air bubbles if required from the finger screw vent on the filter. Tighten the vent. 
  27. Start the stream again. The stream should be aligned in the centre of the waste catch. If needed adjust. 
  28. Close the sort chamber door. 
  29. Open the front cover of the BSC hood and close the front of the ARIAIII lid. 
  30. Close the front cover of the BSC.
  31. Inspect the stream for stable droplet generation, symmetrical droplets and similar breakoff position to the last time the stream was started. Click the sweet spot button and inspect the stream. Allow stream to stabilise. Adjust frequency and amplitude as needed with sweet spot off, updating sweet spot values if necessary.
  32. Carefully check the instrument for wet areas indicating any leaks in the tubing or failing valves.
  33. Do not continue the sort with an unstable stream or leaks. 
  34. Allow ARIAIII stream/lasers to warm / stabilise for at least 30 min.

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  1. Ensure any disinfection solutions (1% bleach, 70% EtOH, 5% Decon, 1:50 Trigene, and any others necessary) are prepared before starting the sorter.
  2. Create a new experiment or duplicate an existing one. Follow appropriate experiment creation workflow, i.e. syntax YYYYMMDD DescriptionSetup in a folder with the following syntax ‘FirstnameLastnameinitial’. Setup experiment using controls provided by the user. Filter controls samples before acquiring on a cytometer if necessary.  Check the FSC-W, FSC-H, SSC-W, SSC-H parameters for recording in the Inspector tab as below to allow identification of doublets.
    Image Removedtab as below to allow identification of doublets. Setup experiment using controls provided by the user. Filter biological samples before acquiring on a cytometer.
  3. Install collection tubes into tube holder and slide into the sort block.
  4. Filter the sample if needed
  5. Load the filtered sample onto machine. Initially run the sample with a low flow rate in the acquisition window and record an appropriate number of events. Draw or adjust gates if needed.
  6. Acquire, record and sort data/cells from each of your samples.
  7. Confirm approval for sorting strategy.
  8. If needed create a new sort layout form the >Sort menu. Input the population being sorted in the Sort Layout window. If doing a 4-way sort, use outer tubes for the lower frequency populations with the higher frequency populations in the middle two tubes. This is to reduce the chance of larger populations contaminating the smaller populations. There are some caveats to this, i.e. number of cells to be sorted, cell size, and side stream stability need to be also considered. Consider appropriate sort mask settings to be used. On the AriaIII, purity or 4way purity modes are commonly used and recommended. 
  9. Install the correct and appropriate collection tubes in the sort collection chamber to collect sorted events. 
  10. Click Sort and turn on agitation to an appropriate value. Adjust flow rate and side streams if necessary. Threshold rate should not exceed approximately ¼ of the frequency to ensure events are spaced out along the stream. Monitor sort efficiency and adjust parameters if necessary.
  11. Avoid running the sample dry to prevent bubbles in the system. If it’s a precious sample add more saline close to the end. When the sample level is low, reduce or halt agitation of the sample to minimise the chance of drawing in air. Monitor sample continuously and stop the sample before it is at a critically low level to prevent drawing air into the sample line.
  12. The sort chamber can only be opened when there is no sample running. Remove collection tube when sort is finished. 
  13. Remove sample from instrument. 
  14. Unless your data should not be synced to the WIMR server, export the data as FCS3.0 files in linear format to D:\BDExport\FCS (as this folder is automatically synced to the WIMR server). If you would like to copy your data elsewhere copy it from this location. Your data should be able to be accessed from the Scientific Platforms networked drive within WIMR.

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  1. Load 10% bleach. Change flow rate to 11 and run for 5 min.
  2. Repeat with 5% Decon 90.
  3. Repeat with DI water.
  4. Stop stream.
  5. If the next sort booked on the instrument in that week uses the same nozzle
    1. Load a full tube of 70% EtOH and initiate clean flow cell command. 
  6. If the next sort booked on the instrument in that week uses a different nozzle
    1. Remove current nozzle. 
    2. Replace with cleaning nozzle.
    3. Load a full tube of 70% EtOH and initiate clean flow cell command. 
    4. When complete - remove cleaning nozzle and remove nozzle clip.
  7. If there is no other sort booked for the week. 
    1. Remove current nozzle. 
    2. Replace with cleaning nozzle.
    3. Initiate fluidics shutdown program from cytometer menu.
    4. Complete fluidics shutdown. 
    5. Remove cleaning nozzle and nozzle clip and replace to default locations.
  8. Spray the sort chamber and other appropriate internal chambers specific to the instrument as well as exposed appropriate external surfaces with 70% w/v ethanol and wipe clean. Spray again with 70% w/v ethanol and allow a contact time of at least 10 minutes.
  9. Wipe the areas and follow with spraying and wiping clean with 70% ethanol
  10. System is now ready to be shut down. Turn off instrument, gas, chiller, AMO & BSC afterwards. Log off computer.
  11. Relieve any pressure from the tanks by venting using the venting valve

Notes

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Detectors

  • Remember there is a range of optimal PMT voltages that will provide similar resolution between dim and negative cells.
  • PMT voltages that are too high will not affect the resolution of dim and negative cells, but can unnecessarily cause spreading of the negative population.
  • Any bright populations with very large CVs (very broad distributions) must have PMT voltages lowered so that the brightest events or MFIs remain within the linear range of the detector.
  • Remember your compensation controls should be representative of your samples, ensuring they are as bright or brighter than your sample (but not log folds brighter preferentially).
  • Remember to leave enough room above the positive signal, and in the linear range of the detector, to allow for increases in fluorescence of certain epitopes.
  • We recommend your samples are filtered beforehand.

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  • Stocking up saline bottles, H2O bottles, gowns, P2 respirators, sterile pipettes, tubes, markers, stationary, reagents and other unlisted as required in the laboratory work flow.
  • Stocking up cleaning solutions including bleach, Decon 90, 1:50 100 TRIGENE & ethanol.
  • Making up cleaning solutions daily
  • Disconnecting the depressurised sheath tank for autoclaving, sheath filter installation, waste tank filter replacement, instrument cleaning and performance checking.
  • Autoclaving autoclavable empty wash bottles holding bleach, ethanol, H20 for sterile H20 / saline and refilling under sterile conditions.
  • Reconnecting fluidic lines as per instrument setup.

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  • Spray the sort chamber and other appropriate internal chambers specific to the instrument with 1:50 100 TRIGENE and wipe clean. Spray again with 1:50 100 TRIGENE and allow a contact time of at least 10 minutes.
  • Wipe the areas and follow with spraying and wiping clean with 70% ethanol.
  • Spray the exposed appropriate external surfaces specific to the instrument with 1:50 100 TRIGENE and wipe clean. Spray again with 1:50 100 TRIGENE and allow a contact time of at least 10 minutes.
  • Wipe the areas sprayed and follow with spraying and wiping clean with 70% ethanol.
  • No further biological samples are to be run on the instrument until maintenance is carried out.

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If the instrument does not automatically shut down the stream upon a stream failure, the stream sample and stream need to be stopped via the software or via the red emergency button located on the instrument. Then after a period of 15 3 minutes, and with the appropriate PPE (gloves, gown, enclosed shoes and safety respirator), the nozzle may be removed if necessary, decontaminated and sonicated if necessary to remove the clog and reinstalled if needed. If the stream failure was due to a clogged sample, the sample may need to be re-filtered or a larger nozzle used. The primary objective is to prevent potentially biohazardous aerosols being exposed outside of the biological safety cabinet. The procedure is outline below.

  1. Ensure BSC is on
  2. Ensure PPE is in use
    1. Gloves, gown, safety glasses & P2 respirator
  3. Stop the stream if the instrument hasn’t automatically. The stream can be stopped via the software or the emergency red button on the instrument
  4. Minimise BSC turbulence
  5. Keep sort chamber closed and sample cover closed to contain aerosols
  6. Increase AMO to maximum flow rate to facilitate the evacuation of aerosols
  7. Allow 15 3 minutes for the AMO & BSC to evacuate any aerosols
  8. Remove collection tubes and sample aseptically.
  9. Spray the sort chamber, sort collection block and other appropriate internal chambers specific to the instrument with 1:100 TRIGENE and wipe clean. Spray again with 1:100 TRIGENE and allow a contact time of at least 10 minutes.
  10. Determine cause of sort failure if possible.
  11. Remove nozzle and sonicate if necessary.
  12. Restart the stream if possible.
  13. If nozzle clog occurred – refilter sample or use larger nozzle before loading sample.
  14. Log the failure in the sort log.

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