BD FACSAria Manual

1Aria-2/4: Turn on Aerosol Management System
Aria-7: Turn on Class 1 BSC by turning Fans on
and set AMS fan to 20%
2The AMS should be operating at 20% at all times
under normal conditions. If clog occurs See Cell
Sorter Clog SOP for further instructions.
1Open Air valve for Aria-2/4. Turn yellow lever on wall until it’s parallel with the pipe.
This is not required for Aria-7.
1
Stream/Sort
QC
Accudrop
FACSAria™
Startup, QC & Shutdown Procedure
Ver.
2019-12-22
Workflow Overview
Daily Startup Procedure for FACSAria™ Sorters
Turn ON
Hardware
Software
Fluidics
&
Stream
Startup
Cleaning
Sort Chamber
Optics
QC
1. Turning On Hardware/Software
2
Post Sort
Cleanup/
Shutdown
AMS On/Off
switch
100% evacuation
button
12
1
2

1Turn ON water bath.
Lauda E100: turn the power switch to on.
Thermo: Press the power button followed by the “enter” button.
3
Lauda E100 water bath Thermo water bath
power button 1st.
enter
on/off
1Restart computer and Login
Username: Administrator or Admin
Password: BDIS
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1Turn ON FACSAria™ sorter.
Once computer is fully on - press the two buttons on the left side
of the FACSAria™. Make sure to turn ON the Main Power first and
then the Laser Switch button.
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Main Power
Laser Switch
1Launch OBIS/Coherent Connection Software
2Aria-2: Coherent Connection. UV Laser typically does not start.
Set to 5 mW and press start. Once started, set to 60 mW
Aria-4: Does not have Laser connection software.
Aria-7: Does not have Laser connection software.
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Coherent Connection on Aria-2.
Look for 355 nm laser and
power to 60 mW.
Check OBIS/Coherent Connection SOP
1Launch Diva Software.
Login name: YourName
Password: ID#
1Wait for connection.
Choose “Use CST settings” in the dialog box that comes up.
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8

2. Fluidics & Stream Startup
1Fill sheath tank and empty waste.
Sheath=1xPBS. To prepare, combine 200 mL of 10XPBS with 1800 mL of dH2O.
For Aria-7:
Tue-Fri: No fluidic startup needed. Remove waste container and add bleach to bring it to a final
concentration of 10% and leave it to the side. Connect separate empty waste container for startup.
Sat-Mon: Fluidic startup is needed, empty the waste into designated EtOH disposal tanks after startup is
completed and connect the empty waste container to the sorter.
For Aria-2/4: at the start of each day empty the waste into the designated EtOH disposal tanks. No
bleach should be added. Fluidic startup is done every day.
1
1Change connections between EtOH tank and Sheath Fluid tank to prepare for sheath
startup.
2
Fluidics setup for EtOH shutdown.
1. Fluidics line hooked up to
designated EtOH filter.
2. Pressure line hooked up to
male connection on EtOH tank.
3. Release valve down, allowing
pressurization of EtOH tank.
Fluidics setup for Sheath startup.
1. Fluidics line hooked up to
designated sheath filter.
2. Pressure line hooked up to
male connection on Sheath
tank.
3. Release valve down, allowing
tank to pressurize with fluidics
startup.
*To Refill tank when empty,
disconnect pressure line and open
release valve to depressurize
sheath tank and fill with 1x PBS.
1
2
3
EtOH tank connected
Sheath tank disconnected
4
5
6
EtOH tank disconnected Sheath tank connected
1Startup fluidics.
Go to Cytometer Fluidics Start-up. Follow on screen prompts.
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1
2
3
4
5
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1Clean interior chamber as well as plates and sort chamber with ethanol.
Make sure all surfaces are dry.
1
3. Cleaning Sort Chamber
Flow cell. Check for fluid or
salt buildup after removing
closed loop nozzle and
before inserting sort
nozzle.
High voltage deflection
plates. Check prior to sort
and clean with a moistened
kim wipe followed with a
dry one to remove any
liquid.
Waste drawer. To clean, fill
the drawer and the center
catch with dH2O to clear
out any potential blockage.
1Sonicate nozzle.
In dH2O, NO more than 60 s.
Closed loop nozzle –for cleaning and shutdown Integrated nozzle –for use during setup and sorting
Tip: While the fluidics startup is
taking place, clean not only the
interior sort chamber but also
all work surfaces on the sorter
and computer table with 70%
ethanol.
1Insert the nozzle into the flow cell.
Remove the closed loop nozzle from the flow cell, verify the o-ring is in place, gently dry with a kim
wipe and place into nozzle holder. Clean the flow cell with a q-tip if any liquid or buildup is seen.
Inspect the nozzle you will be using for your experiment to verify the o-ring is in place and there is no
clog, and insert it into the flow cell with the o-ring facing up.
2
3

1Inspect sheath filter for air bubbles.
If there are any bubbles, bleed the filter after fluidics startup while the tank is pressurized. Be
cautious, as the tank is at high pressure and the bleed valve will need to be slightly loosen to
ensure it does not come off.
4
Sheath filter with
observed air bubble.
Bleeder
valve
1Start the stream.
Ensure the correct nozzle size is shown in stream window. Start the stream by clicking the red “X”
at the top of the stream window.
Note: If incorrect nozzle size shown See FACSAria™ Nozzle Swap SOP for further instructions.
5
1Note: Remaining air in the sheath filter can cause
instability in the stream during setup and through
your run.
6Check the stream image and droplet formation.
Confirm with images below to ensure that the droplet formation is normal.
https://www.bdbiosciences.com/documents/BD_FACSAria_II_User_Guide.pdf
Note: When adjusting the settings
for the droplet formation, never
change the frequency or the gap
that is preset in Diva. These values
are optimized for each nozzle on
each instrument. Only the
amplitude and the drop 1 should
need minor modifications to obtain
optimal droplet formation.
1Turn on the Sweet Spot.
Click the button at the top of the Stream window
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off on

1Record 5,000 events.
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1Create a new tube and name it with the date: yyyymmdd
2
4. Optics QC
1In Diva, go to QC Folder and open the 3-Peak Beads Experiment
1
1Run the CS&T beads in the 3 peak experiment
To prepare CS&T beads, add one drop of BD CS&T beads, Cat#655050, into 350uL of 1xPBS.
(Refrigerate after use.)
3
1Verify that all channels have 3 peaks and brightest peak is falling within target.
For each laser, the primary fluorescent parameter should have an %rCV below.
4
Open 3-Peak Beads
Experiment.
Verify in the
statistics box that
the bright peak on
the primary
channel for each
laser is below 6.0
5. Stream Sort QC and Accudrop
1Verify quality of charging the stream.
Turn on voltage and press Test Sort

3Check Fanning.
Adjust 2nd, 3rd and 4th drop in order to minimize any fanning.
Note: Fanning can cause sort impurity.
2nd drop is not optimal. Fanning of center stream seen. 2nd drop optimized. Tight center stream
2Optimize Accudrop laser diode.
Minimal adjustments should be needed on the laser diode adjustment stage to get the brightest
illumination of the streams.
4Open Accudrop Experiment in QC Folder See FACSAria™ Drop-Charge Delay SOP
Select the tube under the specimen and open the Drop Delay Sort Layout under the Global Sheet.

5Check Sort Precision.
Precision should be at “Fine Tune” in 2 Tube mode.
“Fine Tune” selected
6Load & Run Accudrop beads.
To prepare Accudrop beads, put one drop of BD Accudrop beads, Cat#345249, into 700uL of 1xPBS.
(Refrigerate after use.) Acquire the beads and run at a flow rate of ~2,000 evts/sec.
7Carry out Accudrop sort.
Click “Sort” followed by “Cancel” when the dialog box comes up. Turn on the voltage manually. Confirm
that there is no fanning and that the Accudrop laser diode is aligned.
8Turn on Optical Filter.
Click “Optical Filter” and then adjust the stream deflection so the sort stream falls within the filter.
9Set Drop-charge Delay See FACSAria™ Drop-Charge Delay SOP for more detailed instructions
Adjust Drop-charge Delay value up and down in the Side Stream window until the left stream is >98%.
Note: Percentages in center stream and left stream should add to 100%
Increase optimal drop-charge delay 1 whole number. Verify that left stream is ≤2%.
Decrease optimal drop-charge delay 1 whole number. Verify that left stream is ≤ 2%.
TIP: Hold down control and press arrow up or down to change values by one whole integer.
10 Prepare for sort.
Adjust side streams for deflection into appropriate collection vessels and begin your experiment.
For plate sorting See BD FACSAria™ Plate Sorting SOP.
Drop-charge Delay

6. Post Sort Cleanup and Shutdown
1Clean the sorter after appointment is complete and check to see if fluidics and/or
cytometer shutdown is necessary.
1
Before 5pm (business weekdays only):
Clean with 2 minutes CLEAN (bleach) at flow rate 11, 2 minutes RINSE (detergent) at flow rate 11,
followed by 2 minutes H2O at flow rate 11 and leave instrument on.
After 5pm (or any time on weekends and holidays):
If someone is booked after your appointment:
Clean with 2 minutes CLEAN (bleach) at flow rate 11, 2 minutes RINSE (detergent) at flow rate 11,
followed by 2 minutes H2O at flow rate 11 and leave instrument on.
If no one is booked after your appointment the instrument must be shutdown:
Aria-2/4: Perform Fluidics Shutdown followed by an instrument shutdown (see below 2-4).
Aria-7: Saturday through Thursday: turn stream off, place closed loop nozzle and perform a clean
flow cell with water followed by instrument shutdown (see below 4); Friday: perform Fluidics
Shutdown followed by an instrument shutdown (see below 2-4)
1Change connections between Sheath Fluid tank and EtOH Fluid tank to prepare for
Fluidic Shutdown.
2
2
3
EtOH tank connected
Sheath tank disconnected
1
1Shutdown fluidics.
Go to Cytometer Fluidics Shutdown. Follow on screen prompts.
Note: When prompted to load a tube of cleaning solution, load a tube of 70% EtOH.
3
1Turn off cytometer.
Turn off the main power switch followed by the laser main switch.
4
Note: Please remember to log out of Diva at the end of your appointment. It is necessary for
accurate tracking of usage and billing.
Fluidics setup for EtOH shutdown.
1. Fluidics line hooked up to
designated EtOH filter.
2. Pressure line hooked up to
male connection on EtOH tank.
3. Release valve down, allowing
pressurization of EtOH tank.
*To Refill tank when empty,
disconnect pressure line and open
release valve to depressurize EtOH
tank and fill it with 70% EtOH.
1
2
3
1Turn off AMS or BSC along with the chiller.
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