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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. Spray the sheath tank lid with ethanol.
19. Unscrew the sheath tank lid completely.
20. 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).
21. Fill sheath tank to the weld mark (~10cm from top of tank).
22. Reseal sheath tank - only holding the screw mechanism to keep fingers clear of the lid. 
23. Empty waste container slowly into the sink under running water. Put 100ml of bleach into the waste tank.
24. Reconnect waste container.
25. 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. 
26. Be ready to change the waste catch position, loosen the screws on both sides of the collection assembly, and then start the stream.
27. Check the stream angle. If the stream flows away from the centre of the waste catch, rotate the sort block to adjust. Tighten screws.
28. Close the sort block and thumb screw it shut.
29. Stop the stream. 
30. Start the stream again. The stream should be aligned in the centre of the waste catch. If needed adjust. 
31. Open the front cover of the BSC hood and close the front of the ARIAIII lid. 
32. Close the front cover of the BSC.
33. 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. 
34. Allow ARIAIII stream/lasers to warm / stabilise for at least 30 min.
35. Adjust frequency and amplitude as needed with sweet spot off, updating sweet spot values if necessary.
36. Carefully check the instrument for wet areas indicating any leaks in the tubing or failing valves.
37. Do not continue the sort with an unstable stream or leaks. 

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