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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.
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7. If a CST mismatch dialog appears always click ‘Use CST Settings’
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8. Open the BCS cabinet.
9. Open the front cover of the AriaIII and prob 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. 
       4. Insert sonicated nozzle with the O-ring facing upwards. Lock the nozzle.
       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.
15. Change the waste catch position if necessary. 
16. Stop the stream. This starts air into the sheath tank which is used to minimise contamination of the sheath tank when refilling. 
17. Unscrew the sheath tank lid completely.
18. Remove the air pressurising line.
19. 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).
20. Connect the air pressurising line promptly.
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 bleed out any air bubbles if required from the finger screw vent on the filter. 
26. Start the stream again. The stream should be aligned in the centre of the waste catch. 
27. Close the sort chamber door. 
28. Open the front cover of the BSC hood and close the front of the ARIAIII lid. 
29. Close the front cover of the BSC.
30. 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 values if necessary.
31. Carefully check the instrument for wet areas indicating any leaks in the tubing or failing valves.
32. Do not continue the sort with an unstable stream or leaks. 
33. 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. 
3. Check the FSC-W, FSC-H, SSC-W, SSC-H parameters for recording in the Inspector tab as below to allow identification of doublets.
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4. Install collection tubes into tube holder and slide into the sort block.
5. Filter the sample if needed
6. 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.
7. Acquire, record and sort data/cells from each of your samples.
8. Confirm approval for sorting strategy.
9. 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. 
10. Install the correct and appropriate collection tubes in the sort collection chamber to collect sorted events. 
11. 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.
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. 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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Purity Check

1. Choose Instrument > Cleaning Modes > Sample Line Back flush. Click Start at prompt a few times to clean the valve, and wait for some time (~10-30sec) to clean the sample line, click Stop.
2. Load a full tube of DI water and change flow rate to 11. Clean the tubing for at least 1 minute or until no sample looking events are displayed. Unload water tube and change flow rate to 2-4.
3. Dilute ~5uL of sorted sample into 100uL of diluent (4 drops of saline) in a new tube. Ensure sample agitator is off. Load the purity check tube and record sample for approximately 30-60 seconds.
4. Between running purity check tubes, perform sample line back flush.

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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.
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12. If a CST mismatch dialog appears always click ‘Use CST Settings’
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13. Check if CS&T (quality control) has been performed for the day on the user log that is displayed on the internet browser. Skip the below step if it has already been completed for the day.
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    1. If CS&T hasn’t been completed, prime the instrument (LSRII/Fortessa/Symphony) and then run H20 for 3 minutes or perform a fluidics startup for the CantoII by clicking
       ‘Menu’ > ‘Cytometer’ > ‘Perform fluidics startup’.
    2. Once complete click
       ‘Menu’ > ‘Cytometer’ > ‘CS&T’. Select 'Check Performance', and ensure the correct bead lot number is selected. Each instrument will have a bead lot number displayed on the front of the instrument. Load CS&T beads from the WRHFlow fridge (normally premade, but can be made with 1 drop from the stock bottle in the flow fridge with 300uL saline in a 5mLs FACS tube – refer to MSDS) and click the run button. For the LSRII/Fortessa/Symphony, run beads on low with the dial 5 full rotations from either end. Take note of CST result. If successful, the instrument is ready to use, and the CS&T window can be closed. If CS&T fails, note the error and report it to flow staff before using.
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14. If you are a new user create a folder per user under your group login using the following syntax ‘FirstnameLastnameinitial’. For example, Elizabeth Potter would create a folder called ElizabethP.
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    Creating a new experiment

15. Create a new experiment under your folder, label the experiment using the following syntax ‘YYYYMMDD Descriptive_Name’.
16. Click ‘Cytometer Settings’.
Check the FSC-W, FSC-H, SSC-W, SSC-H parameters for recording in the Inspector tab as below to allow identification of doublets.
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Set up, create and calculate compensation for multicolour experiments
17. Click ‘Menu’ > ‘Experiment’ > ‘Compensation’ > ‘Create compensation controls’.
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18. Leave the compensation tube labels as ‘Generic’ (unless you are using multiple panels in the same experiment as FACSDiva can calculate multiple compensation matrixes and match them with listed fluorochromes, WRHFlow recommends 1 experiment:1 fluorescent panel to avoid confusion). Ensure you have ‘Include separate unstained control tube’ checked (even if you are not using a universal control - as this provides a template displaying all detectors and can be removed later). 
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19. Delete unused parameters from the list of fluorophores to be compensated.
20. Click OK when complete.
21. In the newly created compensation control folder, activate the ‘Unstained Control’ (as it has a useful default specific workspace that shows all instrument parameters).
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22. Acquire any 1 of the compensation controls on low. Adjust FSC and SSC voltages to bring populations onto scale (take note of the FSC & SSC voltages needed to bring bead/cells on scale)
23. Sequentially acquire (*but do not record*) all the compensation controls, while the unstained tube is activated, ensuring for each channel that:
    1. Fluorescence signal is not off scale, ideally less than 2x10⁵ – if off scale reduce the parameter voltage from the cytometer settings window (shown below)
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    2. Compensation control signals are higher than a fully stained sample – as if this isn't the case will result in an incorrect compensation matrix
24. After the above steps, your voltages should now be set. There are other methods to determine optimal detector voltages. Below is the BD method for determining the minimal (and not optimal) voltages that should be used. 
    1. • Acquire unlabelled cells. Adjust FSC and SSC voltages as needed to bring populations onto scale when looking at a FSC/SSC bivariate plot (take note of the voltages needed for cells).
       • Move the already drawn gate, P1, around the cells of interest. Right click a bi-variate plot and click ‘show population statistics’. Edit the statistics display to show the rSD for each fluorescent parameter used (see figure below). Record enough cells to determine the rSD of P1 for all parameters that will be used. You need to record a small file in order to accurately determine the rSD of the unlabelled cells.
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       • Adjust PMT voltage to bring the rSD of the unlabelled cells to 2.5x the value of the rSD of electronic noise (that can be determined from the instrument configuration sheet) that accompanies each cytometer. You need to record a small file in order to accurately determine the rSD of the unlabelled cells each time.
       • Take note of these PMT voltages. These are the minimum PMT voltage that should be used for each detector for optimal detection of dim epitopes.
       • At this point you now have the minimum voltage for each detector needed to separate dim from negative signal - this voltage is not the voltage you should, but provides you with information on the lower bound of the detector voltage.This is the BD method for users wanting to know the minimal voltage that should be used (the voltage used should be higher than the minimal voltage).
25. Now that your voltages are set, you need to determine whether you are using a universal negative or not for compensation. If you are, then you can now record compensation controls - skip to step 20. Otherwise
    1. Click ‘Menu’ > ‘Experiment’ > ‘Compensation’ > ‘Modify compensation controls’. If you are using samples to compensate that have different levels of autofluorescence, we cannot use a universal negative. At this point, before you record the compensation controls uncheck the ‘Include separate unstained control tube/well' option.
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       Bring up the compensation control window.
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       Uncheck the universal negative option.
26. Now we will acquire the samples that will be used to calculate compensation. 
27. Activate the correct tube by clicking the arrow to the left of the tube. 
28. It is okay to change FSC and SSC detector voltages to those noted as you acquire different particles for your compensation controls.
29. Increase the events to record above the default number of 5000 events. We recommend at least 40000 events for a 6 colour experiment.
30. Gate the positive beads of interest for each fluorochrome as P2 as you acquire and record all the fluorescent compensation controls. If not using a universal negative (After recording each compensation control, create the positive and negative gate, by drawing a new P3 gate, as the software looks for a positive signal in the P2 gate and a negative signal in the P3 gate (see below)).
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31. Calculate compensation by clicking
    ‘Menu’ > ‘Experiment’ > ‘Compensation’ > ‘Calculate compensation’, then apply link and save
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32. You can leave the default compensation name or you can give the compensation settings a name following the syntax ‘YYYYMMDD_Descriptive_Name_Comp’
    

    Acquiring your samples

33. Create a new specimen in the same experiment and name relevant tubes with sample and control identifying information, additionally this information can be incorporated as metadata that is useful in offline analysis using the experiment layout menu item from the Experiment menu.
34. Modify the experiment layout to add all the names of the antibodies used for staining to the channel names. Modify the experiment layout to ensure you are recording and storing the correct number and set of events.
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35. Activate the global worksheet (top left bottom on the worksheet toolbar).
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36. Create an approximate gating strategy in the worksheet by drawing some initial plots to view data, the following are recommended to be shown if applicable with/out gating:
    1. Cells of interest (FSC-A x SSC-A)
    2. Single cell gate (FSC-A x FSC-H)
    3. Live dead gate (removal of dead cells)
    4. Fluorophores of interest (fluorophores can be displayed on a bi-variate plot).
    5. Time plot (TIME x last fluorescent parameter used)
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37. Ensure that all fluorescent parameters are displayed in Log, and have bi-exponential display turned on from the Inspector window (unless linear signals are needed).
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38. Activate the tube in the browser by ensuring there is a green arrow next to the tube you are to acquire and record data from.
39. Load your first sample to be acquired onto the instrument SIP. The CantoII and LSRII/Fortessa/Symphony have different setups.
    1. CantoII – move the sample adapter arm to the left, load tube, release sample adapter arm, then click acquire sample - be careful of a pinch hazard from the sample arm. 
    2. Fortessa/LSRII/Symphony – move the sample arm, remove the existing tube (if applicable, from the SIP), SIP can be washed by acquiring H20 between samples to minimise cross contamination, load the next tube and move the sample arm back under the tube.
40. Acquire data on low initially to adjust the FSC / SSC detector voltages to those noted for cells.
41. Now you can run and record your samples with an appropriate flow rate (the instrument electronics can handle ~20000 events per second). It is recommended that due to baseline restore the same flow rate is used for all test samples to be acquired. 
42. Acquire and record data from each of your samples.
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 export folder can be easily accessed from the quicklaunch toolbar. Your data should be able to be accessed from the Scientific Platforms networked drive within WIMR, and via a username/password for non-WIMR users if desired (email wrhflow@sydney.edu.au to setup).
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Cleaning the instrument

43. Clean the fluidics on the instrument by running the machine with 3 min 10% bleach, 3 min 5% decon & 3 min H2O on high.
44. Close the software.
45. Check to see whether you are the last user for the day. If you are the last user for the day turn off the instrument. For CantoII, if you are the last user of the week perform a fluidics shutdown.
46. Log out of the instrument PC, complete the user survey.
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47. Empty the flow cytometer waste container. Add 100ml of bleach into waste tank and refill the sheath. Bleached waste is poured down the sink gently under a running tap, minimising splashing. All other laboratory biohazardous waste is to be removed via the biohazardous waste disposal bins located in the lab.
48. Clean up and decontaminate the area with 70% ethanol.
49. Turn off the instrument using the green power button if you are the last instrument for the daya contact time of at least 10 minutes.
50. Wipe the areas and follow with spraying and wiping clean with 70% ethanol
51. System is now ready to be shut down. Turn off instrument, gas, chiller, AMO & BSC afterwards. Log off computer.
52. Relieve any pressure from the tanks by venting using the venting valve.

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   Data management

• 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@sydney.edu.au and it is the users responsibility that all ethics approvals are complied with. 

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1. Australian/New Zealand Standard - Safety in Laboratories Part 3: Microbiological safety and containment (AS/NZS 2243.3:2010)
2. Australian/New Zealand Standard - Management of clinical and related wastes (AS/NZS 3816:1998)
3. University of Sydney Safety Health & Wellbeing: Guideline for the Decontamination of Clinical/Biological Waste and Spill Management, http://sydney.edu.au/whs/guidelines/biosafety/decontamination_guidelines.shtml#2.2.1.
4. “A Guide to the WIMR Tissue Culture Facilities” – WIMR laboratory guideline
5. BDFACS ARIA III User Manual – Scientific Platforms Network Drive
6. Australian/New Zealand Standard - Safety in Laboratories Part 3: Microbiological safety and containment (AS/NZS 2243.3:2010)
7. Australian/New Zealand Standard - Management of clinical and related wastes (AS/NZS 3816:1998)
8. University of Sydney Safety Health & Wellbeing: Guideline for the Decontamination of Clinical/Biological Waste and Spill Management, http://sydney.edu.au/whs/guidelines/biosafety/decontamination_guidelines.shtml#2.2.1.
9. “A Guide to the WIMR Tissue Culture Facilities” – WIMR laboratory guideline
10. Holmes KL, Fontes B, Hogarth P, et al. International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2014;85(5):434-453. doi:10.1002/cyto.a.22454.
11. MSDS - CST beads
12. WIMR – Management of Hazardous Materials Policy & Procedure
13. WIMR – Critical Risk Management Plan for biosafety
14. WIMR – Critical Risk Management for Waste Management
15. WIMR – Critical Risk Management Plan for chemical safety+
16. OGTR Guidelines - http://www.ogtr.gov.au/

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