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This document describes the operational setup and operation for the BD FACSAria III flow cytometer, a high-speed fixed-alignment benchtop cell sorter located at the Westmead Research Hub. This document includes starting up the system, setting up the stream, checking cytometer performance with Cytometer Setup and Tracking (CS&T) beads, sorting, cleaning, and shutting down the system. It also includes safety considerations and emergency procedures. 

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.

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Monday to Friday

8.00am to 8.00pm

Weekends and , public holidays and shutdown periods. 

Training and Competency Requirements

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List of hazards and risk controls as per risk assessment

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risk assessment

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Task or scenario	Hazard/s	Associated harm	Existing risk controls	Current risk rating	Additional risk controls?	Residual risk rating?
Instrument operation	Electrocution	Contact with electricity can cause electric shock and burns	Routine instrument maintenance - to ensure instrument is in good condition and cabling is not damaged. Electrical equipment annual testing. Educate users to check for visible liquid leaks. Safety circuit breakers and fuses on instrument to prevent general electrocution due to instrument failure, especially in the presence of liquid. Emergency power off button located in laboratories to disconnects power to the red power points and not the blue uninterruptible power points. Routine maintenance - to ensure instrument is in good condition Bright LED - lit to notify of high voltage deflection plates - educate users about importance of ensuring high voltage is off before accessing plates and surrounding area Instrument covers - when sorting all covers are to be in place as physical access is restricted High resistance installed on HV plates - to limit current draw Circuit breakers and fuses on instrument - to prevent general electrocution due to instrument failure, especially in the presence of liquid	low
	Contact/exposure with biohazardous materials	Exposure to biohazardous material can cause health issues PPE while emptying waste tank and adding bleach to waste tank (bleach decontamination of waste material). Engineering control - SIP sheath cover installed to prevent dripping from LSRII and Fortessa in addition to high walled drip tray. Ensure users and support technicians are familiar with risk assessment and SWP for the material used. PPE – gloves, gown & enclosed shoes (P2 mask and safety glasses in sort sort failure). Users empty waste after completion of operation) with running water gently down the sink. 100ml of bleach is added to the instrument waste container after emptying the waste. Biological spill kit - Access to emergency biological spill kit and/or cleaning equipment. Bleach / decon decontamination of sample lines. Project approval process.  Handling samples (e.g. transferring, pipetting) in biological safety cabinet. BSC - Instrument is inside a biosafety cabinet	Software - Software controls to maintain a Electrostatic droplet cell sorter creates thousands of drops per second. Droplets can be small and readily enter airways. Therefore containment of aerosols is critical. This is achieved via having the cell sorter in a BSC, operating with covers, ensuring instrument aerosol management option is properly utilised and following emergency procedures in the case of sort stream failure.  BSC - Instrument is inside a biosafety safety cabinet for increased protection. Software - Software controls to maintain a stable stream Filtration - Ensure samples are filtered prior to loading on the instrument, avoid blockage to minimise aerosol generation during sorting Visual check - Check sample for visible clumps that can cause nozzle clogs Signage - Emergency sort failure procedure in SWP and in room Signage - Signage on door during sorting to prevent unauthorised access during sort.  Aerosol management option installed	low	Manual handling (i.e. lifting, transferring) heavy weight such as waste tank	Manual handling can result in injuries of the back, neck, shoulders, arms or other body parts	Providing information and training to workers on manual handling tasks and request for assistance options Maximum possible weight for tanks is <10kg Trolley/pallet jack - to transfer more than 1 box of saline/water – lifting only 1 box at a time Education - Providing information and training to workers on manual handling tasks Planning - Organising manual handling tasks in a safe way, with loads split into smaller ones, and proper rest periods provided	low	Failure to adhere to SWP	Exposure to laser	High power lasers used in instruments can cause skin/eye damage/burns	Use laser safety shielding at all times - to prevent avoid laser exposure. Do not disengage automatic shutters – electronically or mechanically activated when certain covers are open. Educate users not to circumvent shutters and to avoid looking into any exposed lasers or reflections as laser light can be invisible. Laser safety shielding - to prevent avoid laser exposure. Laser safety shielding - to prevent laser exposure Automatic shutters – pressure driven or mechanical when cover is open	low	Lifting high throughput sampler (HTS)	Manual handling – lifting heavy item	Could lead to injuries of the back, neck, shoulders, arms or other body parts and in use as per SWP.  PPE while emptying waste tank and adding bleach to waste tank (bleach decontamination of waste material). Engineering control - SIP sheath cover installed to minimise aerosol exposure from sample upon unexpected sample unload. Ensure users and support technicians are familiar with risk assessment and SWP for the material used. PPE – gloves, gown & enclosed shoes (P2 mask and safety glasses in sort failure). Users empty waste upon setting up of instrument with running water gently down the sink. 100ml of bleach is added to the instrument waste container after emptying the waste. Biological spill kit - Access to emergency biological spill kit and/or cleaning equipment. Bleach / decon / ethanol decontamination of sample lines. Project approval process.  Handling samples (e.g. transferring, pipetting) in biological safety cabinet. Running the sample dry can introduce air bubbles resulting in unstable stream and rogue aerosol formation. SWP states to not run the sample dry.	low/medium	air bubble detector in sample line to prevent running sample dry.
	Manual handling (i.e. lifting, transferring) heavy weight such as waste tank	Manual handling can result in injuries of the back, neck, shoulders, arms or other body parts	Providing information and training to workers on manual handling tasks and request for assistance options Maximum possible weight for tanks is <10kg Trolley/pallet jack - to transfer more than 1 box of saline/water – lifting only 1 box at a time Education - Providing information and training to workers on manual handling tasks and procedures to minimise manual handling strains - including bending knees and not bending back. Heavy item – lift with colleagues label and education that users can ask for assistance to move the HTS if it is too heavy for them. After hours security can also be contacted. After hours security can be contacted for assistance in lifting if necessary.  Planning - Organising manual handling tasks in a safe way, with loads split into smaller ones, and proper rest periods provided	low
Failure to adhere to SWP	Exposure to laser	High power lasers used in instruments can cause skin/eye damage/burns	Use laser safety shielding at all times - to prevent avoid laser exposure. Do not disengage automatic shutters – electronically or mechanically activated when certain covers are open. Educate users not to circumvent shutters and to avoid looking into any exposed lasers or reflections as laser light can be invisible. Laser safety shielding - to prevent avoid laser exposure. Laser safety shielding - to prevent laser exposure Automatic shutters – pressure driven or mechanical when cover is open	low
Operating AriaIII – pinch hazard	Manual handling – pinch hazard while sample loading	Injury to hand	Education – Providing information, demonstrating and observing proper instrument use while loading samples on the CantoII ARIAIII during training to avoid pinching. Including closing of cover before loading of sample occurs.  Instrument hardware monitoring for failed tube load. Physical cover - Cover to prevent accidental contact with the stage during sample load up Emergency stop button – easily accessible button to stop instrument in an emergency situation	low	Handling hazardous chemical emergency situation	low
	Manual handling – pinch hazard while unscrewing/screwing tank	Injury to hand	Education – Providing information, demonstrating and observing proper instrument use while loading samples on the ARIAIII during training to avoid pinching. Including removing lid completely before filling tank and removing air supply line while depressurising lid for removal.	medium	Removal of air line until lid is being removed.
Handling hazardous chemicals including Bleach, Decon/Contrad, Ethanol, CST beads (Sodium Azide)	Hazardous substance exposure	Eye exposure causing eye damage Contact with skin can cause irritation or burn	Safety goggles are provided for researchers and recommended for use in the lab. Emergency showers & eye wash stations available in shared J.2.06 laboratory. Chemical spill kit available in shared lab J.2.06. PPE – gloves, gown & enclosed shoes are necessary for working in the laboratory. SDS available to users to ensure awareness of relevant chemical hazards and emergency procedures. Required to read and understand before using chemicals.	low
Sample handling	Contact with bio-hazardous material	Exposure to bio-hazardous material	Handle samples in biosafety cabinet PPE – gloves, gown & enclosed shoes are necessary for working in the laboratory. Access to emergency biological spill kit and materials to clean up spills.	low
High pressure gas	Physical injury caused by disconnected tube supplying high pressure air	Tubing connectors may degrade resulting in escape of air	Auto shutting off connections if disocnnected. Emergency shut off valves - Education of user to the location and operation of the emergency shut off valve. Venting ports - Education of users to depressurise any tank/fluidics line before accessing or opening the sheath tank and other components. ·	low
Exposure to sodium azide while performing CST	Exposure to toxic chemical	Acute toxicity	Introduce relevant hazards with SDS. Ensure PPE, i.e. Gloves are worn while handling the sample. Sodium azide is used for a minimal time during the quality control procedure.  Ensure only minimum quantity (1 drop in 300uL) made each time quality control solution is made. Dual barrier protection (gloves, tube) & lid.	low	usage of nitrile gloves if possible - added protection	low

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1. BD – Beckton Dickinson
2. DI water – Deionized water
3. EtOH – Ethanol
4. Sheath solution – saline, 0.9% NaCl
5. SIP sheath - outer SIP steel cover
6. BSC – Biological safety cabinet (class 2 or above)
7. CST Beads – Cytometer Setup and Tracking Beads
8. SIP - Sample injection probe
9. FACS - fluorescence activated cell sorters sorting 

List of resources 

(including personal protective clothing, chemicals and equipment needed)

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• Samples can only be run on the fluorescence activated cell sorters occur after the approval of an associated project in PPMS.
• 100mL of 12.5% sodium hypochlorite (undiluted from the provided containers) must be added to the emptied waste containers at the start of the sorting session.
• All biological samples require filtering prior to acquisition on the FACS to prevent aerosol generation issues arising from clogsSheath tank must be filled prior to stream startup to prevent cell sort failure midrun. 
• Fixed Biological samples may be filtered immediately before acquisition using the WRHFlow provided 50um filters and plastic tweezers. Unfixed samples may be filtered before acquisition using the WRHFlow provided 50um filters or the tube cap filters and plastic tweezers in a biological safety cabinet.can only be run on the fluorescence activated cell sorters occur after filtration. 

Procedure (Step by step instructions for undertaking the task)

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Starting up the FACSAria III system

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 the correct provided credentials. 
4. Log into the PPMS account with your provided credentials. 
5. BD FACSDiva should launch automatically . Once logged 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’
   Image Added
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.
Setting up the stream
11. If nozzle was changed, also change nozzle size settings in the software by setting the correct cytometer configuration. If the nozzle wasn't changed, ensure the correct settings are applied in the software. 
12. Open the sort block door, click the stream start/stop button to turn on the stream.
13. 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.
14. 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.
15. Close the sort block and thumb screw it shut
    1. If it isn't 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.
16. Prop the ARIAIII flow cell cover open, have the sort chamber open and ensure BSC is closed. 
17. Be ready to change the waste catch position and then start the stream. 
18. Change the waste catch position is necessary. 
19. Stop the stream. This starts air into the sheath tank which is used to minimise contamination of the sheath tank when refilling. 
20. Unscrew the sheath tank lid completely. Relieve any pressure from the tanks by venting using the venting valve, and breaking the air-pressure seal by pushing down. Ensure you keep fingers clear of the lid.
21. Fill sheath tank to the weld mark (~10cm from top of tank) and reseal sheath tank.
22. Empty waste container slowly into the sink under running water. Put 100ml of bleach into the waste tank.
23. Reconnect waste container.
24. 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. 
25. Allow ARIAIII stream/lasers to warm / stabilise for at least 30 min.
26. Ensure the computer is on and then log in to the administrator or user account. 
27. If a CST mismatch dialog appears always click ‘Use CST Settings’
    Image Removed
28. 1. 1. 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. 
       2. Open the sort block door, click the stream start/stop button to turn on the stream.
       3. 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.
       4. 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.
       5. Close the sort block and thumb screw it shut.
    2. If the nozzle wasn't changed, ensure the correct settings are applied in the software by looking at the window title bar. 
29. The ARIAIII flow cell cover should be open.
30. Open the sort chamber (we need to view whether the stream is aligned or not. 
31. Close the front cover of the BSC.
32. Be ready to change the waste catch position and then start the stream.
33. Change the waste catch position if necessary. 
34. Stop the stream. This starts air into the sheath tank which is used to minimise contamination of the sheath tank when refilling. 
35. Unscrew the sheath tank lid completely.
36. Remove the air pressurising line.
37. 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).
38. Connect the air pressurising line promptly.
39. Fill sheath tank to the weld mark (~10cm from top of tank).
40. Reseal sheath tank - only holding the screw mechanism to keep fingers clear of the lid. 
41. Empty waste container slowly into the sink under running water. Put 100ml of bleach into the waste tank.
42. Reconnect waste container.
43. 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. 
44. Start the stream again. The stream should be aligned in the centre of the waste catch. 
45. Close the sort chamber door. 
46. Open the front cover of the BSC hood and close the front of the ARIAIII lid. 
47. Close the front cover of the BSC.
48. 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.
49. Do not continue the sort with an unstable stream.
50. Carefully check the instrument for wet areas indicating any leaks in the tubing or failing valves.
51. Do not continue the sort with an unstable stream or leaks. 
52. Allow ARIAIII stream/lasers to warm / stabilise for at least 30 min.

Checking Cytometer Performance with CS&T beads in DIVA.

1. Uncheck sweet spot. Select Cytometer > CS&T.
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. Ensure tube is labelled appropriately. ) 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 alignment is ready right and good 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.
   
3. Select performance check. Ensure correct bead lot CST beads are loaded onto the instrument (Correct bead lot is displayed on asign on the instruement) and click run.
4. If the beads fail, inspect the report and determine whether the stream and sample are as expected. If CS&T continues to fail call BD service.
5. If QC passes, view report to check any warnings.
6. Quit the CS&T window. Click use CS&T settings in Diva.

Checking drop delay (the time between the cytometer seeing an event and the event going into the last connected droplet) with Accudrop beads

1. Prepare Use prepared or if needed prepare Accudrop beads by adding 1 drop of beads into ~250ul of saline in a 5mL FACS tube.  Diluted beads are OK to use within ~1 week. Ensure tube is labelled appropriately. 
2. Open the Accudrop experiment template. Bring out sort window from global worksheet.
3. Load Accudrop beads. Click sort, then click cancel on the dialog that appears to keep the waste drawer closed.
4. Turn on high voltage plates.
5. Make sure sweet spot is on and the stream has been optimised. Using Accudrop beads, toggle optical filter on and adjust the Accudrop laser focus to obtain the brightest bead spot on the side stream.
6. Adjust drop delay to give the cleanest side streams by incremental increases/decreases.
7. You may need to increase / decrease sample flow rate. You may need to sweep the accudrop laser to intensify the accudrop beads visible on the camera. 
8. Check, adjust if necessary and type in drop breakoff and gap values. Drop delay is now set.
9. Unload Accudrop beads.
10. Turn on test sort. Check if the side streams are clean and directed to indicated marks on the screen (i.e. going into the collection tubes intended to be used for the sort). If the side stream is slightly out of the centre of the collection tube, use the slider controls to adjust the side stream voltage. Turn off test sorts and high voltage plates.

Sorting

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. Setup experiment using controls provided by the user. Filter controls samples before acquiring on a cytometer if necessary.
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.

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1. Confirm approval for sorting strategy.
2. 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. 
3. 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.
4. 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.

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 ~5ml ~5uL of sorted sample into 100ml 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.

Data export

1. Export FCS files & batch analyse samples when appropriate into default locations. Move the batch analysis into the FCS files folder ready for syncing or user transfer as setup for data management.

Daily shutdown of instrument

1. Load 10% bleach. Change flow rate to 11 and run for 5 min.
2. Repeat clean with 5% Decon 90.
3. Repeat clean 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
   . Repeat.
6. Remove nozzle and nozzle clip and replace to default locations.
7. Spray the sort chamber and other appropriate internal chambers specific to the instrument with 1:50 TRIGENE and wipe clean. Spray again with 1:50 TRIGENE and allow a contact time of at least 10 minutes.
8. Wipe the areas and follow with spraying and wiping clean with 70% ethanol
9. Spray the exposed appropriate external surfaces specific to the instrument with 1:50 TRIGENE and wipe clean. Spray again with 1:50 TRIGENE and allow a contact time of at least 10 minutes.
10. Wipe the areas sprayed and follow with spraying and wiping clean with 70% ethanol
11. System is now ready to be shut down. Turn off instrument, gas, chiller and hood afterwards. Log off computer.
12. Relieve any pressure from the tanks by venting using the venting valve.

Initial instrument startup, initialisation, setup & quality control

1. If a PPMS booking is made before 8:00am, WRHFlow will endeavour to have the instrument warmed up and ready to use by 9:00am. All instruments are booked from 8:00am until 9:00am for quality control procedures. However if the instrument is not on, turn on the flow cytometer by pressing the green power on button and allowing the lasers to warm up for 30 minutes.
2. Log on to the instrument computer with your WIMR active directory account.
3. Instrument acquisition software (BD FACSDiva) will automatically launch along with an internet browser by default showing the instrument user log and the booking schedule. 
4. Once Windows has loaded and FACSDiva has started, log in to FACSDiva with your group account from the drop down list. The password is the same as the group name in small caps
   1. . 
5. 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.
6. 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.
7. 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.
8. Wipe the areas and follow with spraying and wiping clean with 70% ethanol
9. System is now ready to be shut down. Turn off instrument, gas, chiller, AMO & BSC afterwards. Log off computer.
10. Relieve any pressure from the tanks by venting using the venting valve.

1. 
   
2. If a CST mismatch dialog appears always click ‘Use CST Settings’
   
3. 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.
   
   
   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.
      
4. 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.
   
   

   Creating a new experiment

5. Create a new experiment under your folder, label the experiment using the following syntax ‘YYYYMMDD Descriptive_Name’.
6. Click ‘Cytometer Settings’.
7. Check the FSC-W, FSC-H, SSC-W, SSC-H parameters for recording in the Inspector tab as below to allow identification of doublets.
   
   
   Set up, create and calculate compensation for multicolour experiments
8. Click ‘Menu’ > ‘Experiment’ > ‘Compensation’ > ‘Create compensation controls’.
   
9. 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). 
   
10. Delete unused parameters from the list of fluorophores to be compensated.
11. Click OK when complete.
12. In the newly created compensation control folder, activate the ‘Unstained Control’ (as it has a useful default specific workspace that shows all instrument parameters).
    
    
13. 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)
14. 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)
       
    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
15. 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.
         
       • 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).
16. 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.
       
       Bring up the compensation control window.
       
       Uncheck the universal negative option.
17. Now we will acquire the samples that will be used to calculate compensation. 
18. Activate the correct tube by clicking the arrow to the left of the tube. 
19. It is okay to change FSC and SSC detector voltages to those noted as you acquire different particles for your compensation controls.
20. Increase the events to record above the default number of 5000 events. We recommend at least 40000 events for a 6 colour experiment.
21. 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)).
    
22. Calculate compensation by clicking
    ‘Menu’ > ‘Experiment’ > ‘Compensation’ > ‘Calculate compensation’, then apply link and save
    
23. 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

24. 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.
25. 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.
    
    
26. Activate the global worksheet (top left bottom on the worksheet toolbar).
    
27. 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)
       
28. 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).
    
29. 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.
30. 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.
31. Acquire data on low initially to adjust the FSC / SSC detector voltages to those noted for cells.
32. 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. 
33. Acquire and record data from each of your samples.
34. 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).
    
    
    
    

    Cleaning the instrument

35. Clean the fluidics on the instrument by running the machine with 3 min 10% bleach, 3 min 5% decon & 3 min H2O on high.
36. Close the software.
37. 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.
38. Log out of the instrument PC, complete the user survey.
    
39. 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.
40. Clean up and decontaminate the area with 70% ethanol.
41. Turn off the instrument using the green power button if you are the last instrument for the day.
    

    Data management

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