Attune startup and shutdown

Quick startup checklist

1. Make sure fluid bottles are full except for waste bottles. If necessary, empty waste bottle and refill with 10% bleach (100 mL for Attune, 200 mL for Cytkick)

2. Fluid lines are all plugged in correctly (especially Cytkick autosampler waste!! should hear double click)

3. Log in and run start up procedure

   1. If the Attune has not been run for > 3 days, run another startup procedure to further flush the lines (don’t shutdown, just immediately hit startup again).

   2. If the Attune has not been run for > 1 week, run Debubble with the debubble solution.

4. Run performance test

   1. Make sure optical filter config is correct (no red-stripes, order is correct)

   2. Verify bead lot number

   3. Put 3 drops of performance beads in 2 mL of focusing fluids (should be ~100 ev/s at first)

   4. If Delta PMT is high (>10-20), check Attune maintenance guide pg. 41

5. Run SIP sanitize to wash beads out

Quick shutdown checklist

1. Place a clean 96 well plate into the autosampler (or reuse an old plate with no salt crusts - remove with water/toothpick if there is).

2. Empty the waste containers and refill with 10% bleach (100 mL for Attune, 200 mL for Cytkick).

3. Refill the shutdown and wash solution bottles if needed.

4. Fill a clean tube with 3 mL of 10% bleach and load it into the tube lifter.

5. Run the following depending on how quickly the next user will be using it:

   1. (DEFAULT) If next user will be using it > 2 hr, run Shutdown - standard (40 min)

   2. If next user will be using it < 2 hr, run Deep Clean - quick (10 min)

   3. If last user of the day, run SIP sanitize with Hellmanex, then run Shutdown - thorough (60 min)

6. Log out of the software but do not close the software or log out of the INSTR-ADMIN Windows user or shutdown will fail.

Initial inspection and startup

Estimated time

30 minutes

1. Visually inspect the Attune.

   1. Check the main fluidics syringe for the presence of a salt ring.

   

   A normal syringe without salt ring. The salt ring would be visible at the bottom of the syringe travel, e.g. near the printed label.

   2. Check the internal and external fluid bottles for any leaks or salt residue. Clean up any salt residue with ethanol and a Kimwipe.

   3. Check that other components are in good order (focusing fluid filters, SIP, etc).

Warning

Always grip fluid bottle and data connections by the connector, e.g. as close to the connection as possible. This prevents stress on both the fluid and data lines!

2. Check that all internal fluidics bottles except waste are reasonably full.

   1. If the waste bottle needs to be emptied, empty it into the sink, and refill with 100 mL of 10% bleach. See Attune operation for definition of 10% bleach.

   2. The focusing fluid bottle can be refilled by carefully moving the focusing fluid bottle forward just enough to be able to take the cap off, without disconnecting the fluid or data lines.

   

   The focusing fluid bottle can be moved slightly out for refilling without stressing the fluid or data line.

   3. If disconnecting fluid bottles, the connectors have disconnect “buttons” that must be pressed before disconnecting the bottle. After pushing the disconnect button, carefully remove the line, gripping at the connector, not the fluid line itself to prevent putting stress on the system.

   4. If a fluid line does not reconnect with a click sound, do not force the connection. Check to make sure the instrument-side connector has not been tripped. If it has, press down on the top connector button to reset.

Visual appearance of tripped and normal connectors.

3. Check that the autosampler focusing fluid bottle is full and the waste bottle is empty.

   1. The focusing fluid bottle has a single click lock.

   2. The waste fluid bottle requires a double “click” for locking purposes.

   3. After emptying the waste bottle, fill with 200 mL of 10% bleach.

4. If the instrument is off, turn on the Cytkick first, wait ten seconds, then turn on the Attune.

5. Remove all plates from the Cytkick.

6. Login to the INSTR-ADMIN user, start the software, then login to your specific software user.

7. Run the startup procedure.

   1. If the Attune has not been run for > 3 days, run another startup procedure to further flush the lines.

   2. If the Attune has not been run for > 1 week, run Debubble with the debubble solution.

Performance test

Estimated time

10 minutes, run before the first flow run of the day.

1. Open the top of the Attune, to check the optical filter setup. Replace any red-stripe filters with their stock filters.

2. Click the performance test button in software.

3. Verify the bead lot # of the performance beads.

4. Add 3 full drops of performance beads to a new, clean tube. Dilute with 2 mL of focusing fluid.

5. Run the performance test. In the first part of the performance test, the events per second should be near 100 ev/sec.

6. After performance test, wash the beads out of the sample loop by running SIP sanitize.

Delta PMT	Status
> 10	Run Deep Clean
> 20	Indicates an issue
> 50	Performance will fail

Important

The bead lot number changes roughly every four years and requires a new set of parameter files to be downloaded.

Bead lot changes also requires a new baseline reference to be set.

Instructions for the install/baseline reference and bead lot data files can both be downloaded from ThermoFisher.

Shutdown

Estimated time

30 minutes total, ~2 minutes hands-on time.

1. Place a clean 96 well plate into the autosampler. If reusing a shutdown plate, make sure there are no salt crusts in wells; if so, they can be removed with a toothpick and water.

2. Empty the waste containers and refill with 10% bleach (100 mL for Attune, 200 mL for Cytkick).

3. Refill the shutdown and wash solution bottles if needed.

4. Fill a clean tube with 3 mL of 10% bleach and load it into the tube lifter.

5. Run the following depending on how quickly the next user will be using it:

   1. If next user will be using it < 2 hr, run Deep Clean - quick (10 min)

   2. (DEFAULT) If next user will be using it > 2 hr, run Shutdown - standard (40 min)

   3. If last user of the day, run SIP sanitize with Hellmanex, then run Shutdown - thorough (60 min)

6. Once the shutdown is started, you can log out of the software (but do not close the software or log out of the INSTR-ADMIN Windows user!).

Note

Hellmanex III is the generic version of the very expensive Attune cleaning solution. The Fisher Scientific version is diluted at a 1:2 ratio.