FACS ARIA SOP

ARIA SOP

Starting up the system (every day except Monday)

DO NOT:

• Do not leave the lid of your sample when you place it on the sample injection tube

• Do not use old water solution during the shutdown procedure

• Do not forget to place back the close loop nozzle in the nozzle rack. It will get squeezed by the door and will need to be replaced by Staff

START UP (except Mondays, which requires special training)

1. Turn on the computer 
    

2. Check and refill fluidics

   1. Decontaminate the lid of the sheath fluids tank (large metal container) with EtOH spraying bottle, let it sit while doing the waste

   2. Empty waste in sink, fill with half gallon of pure bleach located under sink

   3. Clean the EtOH off the lid with KIMWIPES, unscrew and open (may need to push harder if the lid is tight), pour sheath fluid in the tank up to the mark, avoid touching anything. close the lid (not too tight)

   4. Disconnect the sheath line and the air line from the EtOH tank and connect them to the sheath tank. Air line = Clear. The Sheath line = Blue. Disconnect only the blue should be connected to the connector under the white sheath filter.

   5. Refill the ethanol tank and write a note that it was done (refilled by XX on XX/XX/XX)
       

3. Starting the Aria to fluidic start up

   1. Turn on the BSC (always leave the BSC on when the Aria is ON)

   2. Turn On the Aria , the main green power  button. If back up compressor connected to the cart =>Turn on the compressor, on the floor (clock or anticlockwise)

   3. Turn on the UV  laser is needed. Open the BD Coherent Connection by right clicking, all laser except UV laser will come up at full power. If UV is needed, go to UV-355 window, click laser stop, then slide the bar down close to 5mW, then click laser start. When you hear the noise of laser starting and observe the pointer goes up to 5mW, slide the bar up slowly to 10-11. After the pointer reaches 10-11, move the bar  up to  20. Minimize the “laser coherent window”

   4. Open Diva by right click and open (Diva will connect to the instrument in ~2min, should be written connecting. On bottom right, you can follow the connection. It should switch from connecting to connected. If it switches to disconnected, it means that the connection did not work and you will have to shut down the computer and turn off the aria. Then, turn on the computer again, then the Aria.

   5. Click “fluidic startup” in cytometer tab and follow the instructions (while waiting you can fill up the sheath fluid bottles as sterile as possible).
       

4. Turn on the Stream

   1. Select the configuration (go to Cytometer, View Configurations, select the configuration Nozzle/laser, click set configuration XXX and click OK). Close the view configuration window, go back to diva. Verify that the configuration is the right one by looking at the nozzle size of the “drop/breakoff  window” (right side of screen) and on top of the Diva Browser

   2. Install the nozzle that you selected

   3. Change the Neutral density filter (ND1 for 70um and 1.5 for all other nozzles)

   4. Start the Stream while sort chamber is open

   5. look at the stream and break off. After 3 minutes the drop should be stable

   6. The stream should fall in the middle of the waste compartment. If not, stop the stream, sonicate and reinsert nozzle. If it is still not in the middle, unscrew the sort block using tools and reposition the sort drawer

   7. Change the amplitude to get the first drop in the right position and the gap appropriate (i.e. usually it is very close to where it was last time the nozzle was used).  If the stream doesn’t look good, stop the stream and sonicate the nozzle (put it in a 50 ml falcon tube with 2 -5 ml water, sonicate for 2 min, Carefully take out from the tube with touching the red O ring. Dry out and put it back.)

       

5. CST Calibration: Run CS&T for laser calibration

   1. Before calibration make sure that the neutral density filter is either 1 (for 70 um) or 1.5 for all other (85 um and 100um). This neutral density filter is located near the “nozzle rack” near the flow cells. Please be careful to not disconnect any wire and tubing from this area. If the 1.5 neutral density filter is in, switch it to the 1 neutral density filter which is in the yellow box on the computer table. To switch filter, you need to remove the nozzle rack and free the close loop nozzle. Once you swap the neutral density filters, place the nozzle rack back and reposition the close loop nozzle  filter on the nozzle rack

   2. Gently close the sort door and the Aria main door

   3. Adjust the camera knob on the Aria in order to have the brightest signal  “sort camera” (horizontal window)

   4. Go to Cytometer and click CS&T (takes a minute to disconnect DiVa and open CST). Note don’t over click. If so, the CST window might disappear under the Diva. (click CST to have it on top)

   5. Take the 5 ml FACS tube containing CST beads from 4˚C (or make it if the tube is empty: 1 ml with 3 drops of CST beads (blue cap)

   6. Place the tube on sample port

   7. Click run in the CST window

   8. Note: The sweet spot need to be off. It is OK if the drop moves during the CST calibration. Also make sure

   9. If CST failed (complete failure), it could be the 1.5 neutral density filter that is in place. Usually it passes but sometime, one of the parameters is not OK, large CV or other. Then proceed but tell flow core staff

   10. Exit CST, click “use CST” on DIVA

        

6. Accudrop for the drop delay calibration

   1. After running the CST, make sure to readjust the amplitude for the drop 1 and the gap. Turn on the sweet spot

   2. Adjust the camera knob on the Aria in order to have the brightest signal “sort camera” (horizontal window)

   3. Take the accudrop solution from the fridge. If the existing accudrop solution run low, prepare accudrop by adding 5-6 drops in 2ml of PBS.

   4. Open Accudrop Expt with proper configuration. If message “no match in configuration” click continue

   5. Open specimen, click on tube (i.e. green arrow to the left)

   6. Open Sort layout in Global Sheet

   7. Place Accudrop drop tube on the port

   8. Click load and Acquire

   9. Run at 1500 events/sec for 85,100 um nozzle and slightly below 3000 events/sec for 70um nozzle

   10. Click successively on “voltage”, “optical filter” and “sort”

   11. Adjust the center left slider (in the “sorting” window) to see the sort beads fall in the quadrant

   12. Select auto delay and run. When exit autodelay, check for 30 sec to make sure the delay autocalibration gave >95%

   13. Verify that the value for the drop delay are similar to one observed recently (excel file on the desktop of the MacBook Pro). Only Flow core staff enters the value, users can use these values as a guide

   14. Note: The vibration of the Aria sometime affects the camera, which makes the quadrant not fitting the position where the beads are. To fix this, open the aria door, gently pull toward you the black block with a silver node toward you. While doing so look at the horizontal window.  The picture of the stream should move to the right. Please if you don’t know what this refers to, find someone who does.

        

7. Sorting and changing tubes

   1. Make sure the stream is properly adjusted so the sorted drops fall into the center of the tubes: To do this, choose the proper adapter (2 tubes, 4 tubes etc.…) and place empty tubes in the slot that you need. Open the sort chamber to see the stream, in DIVA go to the sort window, turn the plates on, click test, and open the drawer, adjust the slider to have the stream falling into the tubes.

   2. Open an existing experiment and duplicate without data or click experiment/new experiment and select a template. Check your samples and record data, adjust the gates. Place your collection tubes into the collection rack. Open the sort layout (in global worksheet, make sure the collection rack corresponds to what you set up, select purity and number of cells- if the maximum cells are needed, select continuous). Acquire sample and if the event rates is 4 time less than the drop frequency, click sort (i.e. 20,000 events/s, for 70 um nozzle, 10,000 events/s for 85 um nozzle 6,000 events/s for 100um nozzle). Make sure to click OK when the window pops and ask you to open the sort drawer.

   3. While sorting, make sure the event rate is constant, the drop is stable, the drawer is open. Check the samples to make sure you still have cells, check the collection tubes to make sure there is enough space to sort the cells.

   4. To change collection tubes:

   5. Pause sort, 

   6. Stop acquiring your sample, 

   7. Slide the collection tube rack 

   8. Change the collection tubes and slide the collection tubes rack back into place

   9. Click acquiring, if events rate stable, click resume

       

8. Clogging

   1. Stop stream (if not already off)

   2. Place the AMO (Aerosol management) to 100% for 1 min, decrease the AMO to 20%

   3. Open Cytometer hood, and sort chamber

   4. Take out the nozzle, place it in a clean 50 ml falcon with 5 ml of clean water, sonicate it for 30 seconds in clean water 

   5. Grab the nozzle with the tweezer avoiding touching the red O ring. Dry it with compressed air 

       

9. Changing nozzle:

   1. Stop stream, wait 1 min

   2. Open Cytometer hood, and sort chamber

   3. Take out the existing nozzle and   replace with correct one.

   4. Go to “view configuration” in the cytometer tab. Select the correct nozzle (e.g. 100 with UV)--Set configuration--OK and OK.

   5. Turn on the stream, adjust the amplitude

   6. Before calibration make sure that the neutral density filter is either 1 (for 70 um) or 1.5 for all other (85 um and 100um). This neutral density filter is located near the “nozzle rack” near the flow cells. Please be careful to not disconnect any wire and tubing from this area. If the 1.5 neutral density filter is in, switch it to the 1 neutral density filter which is in the yellow box on the computer table. To switch filter you need to remove the nozzle rack and free the close loop nozzle. Once you swap the neutral density filters, place the nozzle rack back and reposition the close loop nozzle  filter on the nozzle rack

   7. Run CST (see section above) or at least perform a QuickCST to make sure the signal is OK

   8. Run Accudrop (see section above)

       

10. WEEKLY SHUTDOWN

    1. Run 10%  Bleach until clean (change tube if necessary)

    2. Run water until no events are seen 

    3. Stop the Stream and wait 1 minutes for pressure to stabilize

    4. Select Cytometer/ Fluidic Shutdown (follow instructions)

    5. Remove the nozzle and insert the close loop nozzle, Click next step

    6. Disconnect the sheath line (blue line to be disconnected under the sheath filter) and connect it to the adapter of the EtOH tank after the filter (yellow line). Note that if done properly there should be only one filter on this newly assembled line. Disconnect the air line from the sheath tank and connect it to the ethanol tank. Click next step. 

    7. Depressurize the sheath tank by lifting the ring with a blue tape. Click next step. 

    8. Fill a new FACS tube (bottom drawer of the tool cart) with 3-4ml of clean water (i.e. from the bottle). The flow cell will sit overnight in this solution, so make sure it is really clean water. Click next until the shutdown is complete

    9. Quit Diva

    10. Turn off the Aria, turn off computer, turn off water bath, turn off the Biosafety cabinet and turn off the back up compressor if it is connected (the switch button is on the opposite side of the gages side)

         

11. WEEK-END SHUTDOWN (when no user are booked on the week-end)

    1. On Friday night, we run ethanol also through the sheath filter to decontaminate the filter. So, instead of disconnecting the lines after the sheath tank, we disconnect the entire blue line including the sheath filter and connect it directly to the ethanol tank. 

    2. On Monday morning, disconnect the sheath line with the filter from the ethanol tank and replace the entire new sheath filter with blue line prepared by the flow core staff. Note that the filter need to be bleed to remove all air bubble.  Refill tanks as usual and perform 3 start up in a raw.

        

12. Advice corner:

    1. Load every 4 drop: i.e. the frequency (2nd number of the breakoff window) divided by 4 and multiplied by 1000 should be your target event rate. 

    2. Take advantage of the threshold. For example, threshold on FSC and SSC to cut off the small debris. This will lower the events counted and therefore you can increase the flow rate to reach the ¼ drop loaded with an event. This will also increase your yield as a drop containing a debris and a cell of interest will be sorted.

    3. Concentrate your cells to avoid increasing the flow rate which increases the dispersion of your population (i.e. the CV). If you have 1 log shift or more, it does not matter. 

    4. Check purity of the sorted cells

    5. Use compensation controls and FMO (fluorescence minus one) for weak markers

    6. To have good purity, don’t be too generous with the singlets gates

    7. Dead cell markers (for example the invitrogen LIVE DEAD markers)