Aria Cell Sorter Start Up SOP

*Between the hours of 9-5pm Monday through Friday, steps 1-20will be completed for you*

  1. Empty wastedown the lab sink if full. Unscrew the w and take to the sink and pour down the drain. Add 1L 100% bleach into the empty waste container.
  1. Refill sheath tank with 1x PBS to the top 'fill line' (Note. Use the autoclaved 1x PBS prepared especially for this instrument. DO NOTuse the NERL Diluent Sheath Fluid that is used for the LSR2). Ensure air and fluid lines are connected to tank.
  1. Turn computer on, login into the Admin account: password BDIS#1.
  1. Open Tera-Term Program from the desktop. You do not need to do anything with the program.
  1. Ensure the flow cell access door is raised and the sort chamber door is open. Youwill want to check the plates to make sure they are dry and clean before you turnon the stream.
  1. Turn on main power switch (Green button-left side) and verify that Tera-Term has connected to the cytometer. Turn on the air compressor in the corner for the Aira 2u (Captain America) and Aria 3.1 (Falcon). The Aria 3.2 (Captain Marvel) and 3.3 (Spider-Man) do not require this step.
  1. Wait 30 minutes forlasers to warm up before running samples.
  1. Turn on aerosol management system if you are sorting a BSL-2 sample (anything human, Aria 2u and 3.2).
  1. Optional: Turn on water bath for cooled sample collection.
  1. Open FACS Diva and log into your account with your Sunet ID and password.
  1. Select Use CST settingsin the CST dialog box. Most samples are run on a70u nozzle. If you use another size nozzle and have set up the stream, go to cytometer>view configurations and switch to the 100uM setting. Close out of the view configurations and CST setting windows to reconnect to the machine.
  1. Ensure the instrument is connected to the computer (Instrument Connected status at bottom of Cytometer window).
  1. Perform Fluidics Start Up (Cytometer>Fluidics Start-up) and follow the prompts.
  1. Select the correct nozzle size for your experiment. We typically use 70uM and 100uM for the cell sorters. If you require other nozzles sizes, please ask the core director.
  1. Turn stream on and wait 1-2 minutes for stream to stabilize. Hit sweet spot to bring to previous settings used for sorting.
  1. Check stream images posted for correct settings on each machine.
  2. Frequencies may vary slightly from one transducer to another, the stream imagesabove each instrument will reflect the correct setting for that machine.

•For 70um approximately 89 kHz, 70psi gap 6-10

•For the 100μm, approximately 30kHz 20psi gap 10-18

  1. Set the drop delay (Accudrops can be found in the fridge by the door on the top shelf).

•Find Corey’s folder and open according the your nozzle size.

•Run Accudrops: (for 70uM nozzle, make sure events/second are between 1500-3000 and for 100uM make sure it is between 500-1500.

•Go to sort> new sort layout. Set precision at initial and population to sort at P1.

•Set window extension to 0 in the laser tab

•Add 1 drop of Accudrops per .5ml PBS. Load Accudrop tube, acquire sample and click sort in the sort tab. Press cancel to have beads go to waste. For 70uM nozzle, make sure events/second are between 1500-3000 and for 100uM make sure it is between 500-1500.

•Adjust drop delay to get percentage of events to be above 95% in the left box of the sort block window. Switch precision tab to fine tune again adjust to 95%. If the stream is stable (above 95% for 10 seconds) unload tube, turn window extension back to 2.0 and place Accudrop tube back in the refrigerator.

  1. Set the side streams to the number and types of tubes that you will be collecting into. Remove tube collector for plate sorting. Check to make sure the streams drop in the center of your tube by turning the voltage on, turning test stream on and retracting the waste drawer. Adjust the side streams accordingly.
  1. You are ready to sort!

***For clean-up and shutdown procedures, please see the plastic sheet by each sorter ***

Common issues/Problems with BD Cell Sorters:

1. Fluidics Start-Up/Shut-down Fails: The most common reason is either the waste is full or the sheath tank is not properly sealed. Empty the waste tank and add 1L of bleach described about for changing the waste. For the sheath tank, make sure you hear no hissing from the sheath tank while fluidics is starting. If you do, unscrew the top of the tank and pull up until you cannot hear hissing. Retighten the tank and you should feel the lid push up tightly against the tank.

2.Cogged nozzle:Place the nozzle in a capped 5ml tube containing DI H20. Place this tube in a waterbath sonicator for 1-5 minutes. View the orifice of the nozzle under 10x zoom to visually verify there are no obstructions to the orifice. Also ensure that the orange o-ring looks OK (no cracks).

3. Stream Break off and Droplet Formation is Unstable: There could be a few things happening here:

A. Check CST configuration matches the configuration you need for your experiment and the proper nozzle is in place

B. Check the pressure gauge on the left side of the wet cart below the sorter. If it is below 90 psi, BD will have to be called.You will likely still run a 100uM nozzle if this happens since it requires a lower PSI.

C. Dense samples can also effect stream stability. Diluting your sample so that you can run 1000 event/sec on the setting of 1 will often lead to better stream stability.

D. Dirty nozzle/flow cells: You can clean the nozzle as described above and clean the flow cell by going to cytometer>cleaning>clean flow cell with 10% bleach

E. Over time, the stream can drift and you will have to readjust manually to get back to your target sweet spot. Check periodically for the best sort efficiencies.

F. If you are not able to get satellite drops to join parent drop before 5 drops, you may have to readjust the frequency slightly. Frequencies are typically best between 87 and 94 khz and each machines is a bit different.

4. Stream camera is obstructed from stream clog or spraying:Take a qtip (found near machine) and clean under the flow cell until you can no longer see the obstruction on the camera.

5. Stream fanning: Typically due to an instable stream or drop maladjustment. Make sure your stream is stable and the Gap is within the desired target value for the nozzle and you have nice drop formation. Check that the 2nd, 3rd and 4thdrop are set at 20-17, 6-10, and 0-3 respectively. Also check that the Gap is set according to the nozzle size and that your stream is stable. Recheck by turning the voltage on and test sort to make sure streams are stable and no longer fanning.

6. High voltage error: There is excessive liquid in the sort block, under the flow cell, or on the defection plates. Turn off the cell sorter (do not vent the sheath tank) and clean the sort block, defection plates, and chamber with ethanol and ensure no liquid or accumulated slats are present. Restart the machine and allow it to reconnect to the computer. Press in the emergency eject button to reset the system. If you turn the machine on and the voltage light is still on, restart the machine again until it no longer activates when it is on. Restart FACS diva and turn the stream on. Test sort with voltage on and see if the error repeats, if it does, restart cleaning from the beginning.

7. Can’t see side streams, waste stream does not hit middle waste chamber: This is due to either the laser being out of synch with the stream, the camera that see the drops has shifted, or the sort block was moved from a previous sort adjustment. Ensure that the stream hits the middle waste chamber. Adjust the micrometer to get the laser to illuminate your streams.

8. No signal is seen when samples are running: Make sure the lid is closed completely. If this does not fix the problem, tun stream off, log out of FACS Diva and restart the sorter manually. Turn everything back on and reconnect to the machine with the lid closed. This should fix the connection issue between the sorter and the computer.

If you are still having problems with the sorters and none of these suggestions can help you, please call Corey Cain at 209-324-9540 or email me Corey at .

Alternatively, you can call BD with your concern at 1-877-232-8995, prompt 2. You will need to provide the serial number of the sorter (found on the green tag on the left side of the machine).

Aria Serial Numbers:

Aria 2u (Captain America) :P99900023

Aria 3.1 (Falcon): P28200111

Aria 3.2 (Captain Marvel) :P64828200017

Aria 3.3 (Spider-Man): P64828200406

May the Sort be with you!

Detailed Instructions for Accesory Items and Experimental Set-up (Adopted from Stanford Flow Cytometry Facility and BD Aria Handbook)

Setting of the drop delay with Accudrop beads:

  1. Click on the Accudrop experiment, found in Corey’s folder. Select the worksheet that matches your nozzle size
  1. Highlight and make active a tube in the experiment
  1. Load Accudrop beads on sample loading arm
  2. Accudrops can be found in fridge by the door. Add 1 drop per 500ul. There are typically already prepared accudrps in the refrigerator, so use those first before making a new tube of Accudrops
  1. In the laser tab in the Cytometer window, lower the Window Extension to 0
  1. Set the Far left, Right and Far right stream deflection values to 0
  1. Use the left deflection stream for a 2 tube sort 7. Turn on the voltage plates and initiate a test sort
  1. Adjust the Accudrop laser focus for the bright spot in the left and center streams
  1. Adjust the left stream to fall in left optical filter window
  1. Adjust the sample flow rate to
  2. 70u 1.500-3,000
  3. 85u 1,500-3,000
  4. 100u 500-1,500
  5. 130u 500-900
  1. Note- if this cannot be achieved using a flow rate setting between 1 and 5, adjustthe bead concentration
  1. Our Accudrop sort layout comes up defined, below are the manual steps forsetting up the layout
  1. In the sort layout click on the blank white tab under “Left” and choose the population that encompasses all events
  1. Select Device> 2 tube, Precision>Initial
  1. Turn on the voltage in the Side stream window. Center stream should not move-if it does clean the plates with Di water
  1. Click the sort button at the lower left of the Sort Layout. (Click Cancel to thewaste drawer comment.) Observe a left deflection stream in the stream window
  1. Click the Optical Filter button in the stream window to allow only the Accudropbead fluorescence to be seen
  1. Adjust the voltage slider in the stream window so that the sorted Accudrop beadsfall in the middle of the left optical filter box
  1. Adjust the Drop Delay incrementally to maximize the percentage of beads in thedeflected stream
  1. Change the Precision setting in the Sort Layout to Fine Tune and maximize again.You ought to be able to get 98-99%% of the Accudrop signal showing up in thedeflected stream. Once the drop delay is set, click the Optical Filter control to move the emissionfilter away from the camera, turn off the deflection plates, reset the windowextension to 2 in the laser tab and unload the tube

Aerosol Containment System for BSL-2/ Human samples

1. When sorting human material or other potentially hazardous samples, the Aerosol

Management System must be used

2. Ensure that the main power button on the back of the Aerosol

Management System Evacuator is on

3. Press the POWER button on the membrane panel of the Aerosol

Management System Evacuator

4. Ensure that the suction control rate is set to 20%.(Do not set the suction control

rate above 20%, higher rates could affect the stability of the side streams.)

5. Verify that the filter flow gauge reads less than 2.4 inches

6. Sort as usual, keeping the sort collection chamber door closed while samples are running

7. Turn off power to aerosol management on power strip

Repeated failure to turn of the Aerosol Management System will result in the lab purchasing a new filter for the VA Flow Cytometry Core.

Experiment Set-up and Compensation

Remember: All samples must be filtered before running on the instruments!

  1. Create a folder clicking the folder button on the top left screen
  1. Add a specimen by clicking on the syringe button
  1. Set up some preliminary dot plots on the global sheet to start adjusting your FSC and SSC detector voltages. Typical FSC can ranges from 100-300 and SSC around 250-400. Each machine is slightly different so do not assume one detector setting will be the same for another machine
  1. Select ExperimentCompensation SetupCreate compensation tubes. This willgenerate a new specimen in your experiment called Compensation Controls.Within the specimen it makes a tube and a worksheet for each compensationcontrol, including one for unstained cells
  1. Run unstained cells first. Set Events to display to 5000
  1. Adjust the FSC and SSC voltages to place the population of interest on scale
  1. Adjust the FSC threshold value to exclude most of the debris without excludingthe population of interest.Remember what you cannot see you cannot exclude from sort so set thresholdscarefully
  1. Move p1 gate on FSC/SSC plot to include your cells of interest, then right-clickand “Apply to All Compensation Controls” if all your controls are expected tofall in the same FSC v SSC gate
  1. Run each sample in the unstained cell tube area so we can define that all eventsare on scale, and that each stain is most dominant in its own channel. Try toavoid lowering voltages below these target values. It is acceptable to have thenegative peak shift above the first log decade in some detectors
  1. Put on your positively stained sample to verify that the positive peaks remain onscale. Lower voltages on those parameters that are off scale. Remember, if yousignificantly lower the PMT voltage below the baseline settings in order to bringthe positive population on scale, then the dim populations might not be easilyresolved from the negative populations for that parameter
  1. Run the compensation samples and collect data for each in the appropriate tube.Use “Next” to move between tubes. For each tube, make sure the integral gateencompasses your positively stained cells
  1. After collecting all the compensation controls, review all the samples to verifythe gating is correct
  2. Select ExperimentCompensation setupCalculate compensation. Thiscalculates a compensation matrix. If the calculation is successful, a dialogappears for you to enter a name for the compensation setup. To keep track ofcompensation setups, include the experiment name, your initials, or the date inthe setup name. Error messages that warn you of >100% compensation valuesindicate that the color combination at the settings you have chosen is not optimal.You can ignore these messages, but it would be best to try increasing ordecreasing voltages on the problem channels and rerunning all your compcontrols at new settings. You will have to recalculate compensation. If there arestill problems, it may indicate that you do not have a good color combination

Cell analysis and Data Acquisition

  1. Make a series of plots on the global worksheet that you will need to set up youracquisition
  1. With the tube icon in the Browser window activated, load your tube and recordthe data
  1. Pull up a population hierarchy and make gates as needed for your sort or analysis
  1. Make sure you visualize the bi exponential display to bring populations off axisand allow gates to isolate all cells
  1. Use the Bi Exponential Editor to adjust when necessary
  1. Save an analysis template so you can reuse the gating structure, click on analysisworksheet, rename it and export to saveRemember to do a sample line backflush between samples

Sorting Samples

  1. Create a new sort layout
  1. In the Sort Layout, select the Device and Precision you require. You can alsoselect a specific number of cells to be sorted into each tube if you like, otherwise,just leave it as “continuous”
  1. Assign the sorting gates to be applied for each deflected stream
  1. Insert a collection device with prepared tubes
  1. Click Acquire and then click the Sort button in the Sort Layout. Click OK to openthe drawer
  1. A sample line backflush should be used between samples and before runningcleaning solutions

Tips for sorting