Date of revision and approval

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The procedures apply to users operating the BD FACSAria III in room J.2.04, level 2 of WIMR. All personnel require training prior to independent operation of the instrument. Training is conducted by a trained operator or the scientific platform manager (if appropriate) with competency demonstration necessary before authorisation of access. Competency is assessed via demonstration of independent instrument operation, in conjunction with verbal explanation of all aspects of operation of the instrument and troubleshooting common and simulated faults. All instrument operation is to be conducted by trained operators. OGTR requirements for safe work in a PC2 laboratory apply.

Hours of operation and emergency contacts

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Advanced Specialist on 0-8627 3216

Cytometry/Imaging/EM Manager on 0-8627 1820

Scientific Platforms Manager platforms Director on 0-8627 3210

Email: westmead.cytometry@sydney.edu.au

For POLICE, AMBULANCE or FIRE emergencies contact 0-000

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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 BSC cabinet.
9. Open the front cover of the AriaIII and 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 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>View Configurations>Select correct nozzle>Set Configuration. 
       6. 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.
    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, loosen the screws on both sides of the collection assembly, and then start the stream.
15. Check the stream angle. If the stream flows away from the centre of the waste catch, rotate the sort block to adjust. Tighten screws.
16. Close the sort block and thumb screw it shut.
17. Stop the stream. This still results in air into the sheath tank which is used to minimise contamination of the sheath tank when refilling. ). 
18. Close the front cover of the BSC.
19. Spray the sheath tank lid with ethanol.
20. Unscrew the sheath tank lid completely.
21. Relieve all pressure from the tank by venting using the venting valve, and breaking the air-pressure seal by pushing down on the lid. Ensure you keep fingers clear of the edges of the lid and remove it. It can be stored temporarily on the fluidics cart (keeping the inner face not touching anything).
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 ventbubbles if required from the finger screw vent on the filter. Tighten the vent. 
27. Be ready to change the waste catch position, loosen the screws on both sides of the collection assembly, and then start the stream.
28. Check the stream angle. If the stream flows away from the centre of the waste catch, rotate the sort block to adjust. Tighten screws.
29. Close the sort block and thumb screw it shut.
30. Stop the stream. 
31. Start the stream again. The stream should be aligned in the centre of the waste catch. If needed adjust. 
32. Open the front cover of the BSC hood and close the front of the ARIAIII lid. 
33. Close the front cover of the BSC.
34. Inspect the stream for stable droplet generation, symmetrical droplets and similar breakoff position to the last time the stream was started. Check for satellite droplet merging typically within 3 drops of breakoff. Click the sweet spot button and inspect the stream. 
35. Allow ARIAIII stream/lasers to warm / stabilise for at least 30 min.
36. Adjust frequency and amplitude as needed with sweet spot off, updating sweet spot values if necessary.
37. Carefully check the instrument for wet areas indicating any leaks in the tubing or failing valves.
38. Do not continue the sort with an unstable stream or leaks. 

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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’. Check the FSC-W, FSC-H, SSC-W, SSC-H parameters for recording in the Inspector tab 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. Ensure the 'Sort in Progress' signage is on the door. When the sort is finished this sign can be removed.
12. 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.
13. The sort chamber can only be opened when there is no sample running. Ensure the waste draw is closed before opening sort chamber. Remove collection tube when sort is finished. 
14. Remove sample from instrument. 
15. 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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• Under no circumstances should there be any patient/person identiable data recorded on the instruments as the data is not privately held and is accessible to all researchers. 
• User data is deleted after 7 days off the instrument computers, it is the responsibility of the user to have transferred data to a secure location.
• WRHFlow recommends exporting data from BD FacsDiva as FCS3.0 files in linear format to D:\BDExport\FCS. This folder is automatically synced to the WIMR Scientific Platforms Drive that all WIMR active directory users can access. For the sync to work properly your experiment must be in a folder with "FirstnameLastinitial" syntax. Please note this folder is accessible to all researchers.
• If you would like your data accessible from outside of WIMR, a user specific share can be setup. This works by creating a copy of a specified folder on university provisioned storage that is shared with the user. This requires an email to wrhflow@sydneywestmead.cytometry@sydney.edu.au and it is the users responsibility that all ethics approvals are complied with. 

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• Spray the sort chamber 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.
• Wipe the areas and follow with spraying and wiping clean with 70% ethanol.
• Spray the exposed appropriate external surfaces specific to the instrument with 1:100 TRIGENE and wipe clean. Spray again with 1:100 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.

Emergency procedures

Emergency procedure folder can be found here in the sample prep lab:

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You can also access all these information via WIMR intranet:

https://intranet.westmeadinstitute.org.au/workingatwmi/HandS/Pages/Sarah%20Johnston.aspx

All emergencies need to be reported to the emergency contacts listed above. Specific chemical exposure procedures are below.

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