TITLE: COPAS wormsorter v.2
AUTHORS: Saul  Moore, Ida Barlow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for dispensing adult worms using the COPAS 500 flowpilot</div></div>

[STEPS]
?. [Prepare equipment]
Turn on equipment in the following ordera.Sorter (left side of unit near bottom)b.Computer and monitorc.Laser, located under computerd.Vacuum pump (switch on wall to right of sorter)
?. [Prepare equipment]
Discard waste contents that are in the recovery cup (small shallow cup on the left-hand side of the machine)a.Check/clean the recovery cup filterb.Replace filter ~once per week with a new cell strainer (it should clip into place)
?. [Prepare equipment]
Check that there is water in the sheath. If the water is low (less than 5cm above the top of the green sensor), fill up withsterile H2O (from 4thfloor kitchen)
?. [Prepare equipment]
Make sure that the recovery cup and sample cup are securely tightened so that there are no leaks in the system
?. [Prepare equipment]
Check that there is sterile water in the sample cup
?. [Prepare equipment]
Check for stray plate lids etc in the area around the COPAS stage and remove
?. [Prepare software]
Open dbgview – should always be running in the background
?. [Prepare software]
Open FlowPilot software:a.File -> Load Experimenti.Select appropriate template experiment from Documents/BehaviouralGenomics (adults, L4s, L2s all have different gates set)b.File -> Load sample (will load the sample with the appropriate gate for your experiments)
?. [Prepare software]
Save your experiment for this day of sorting (YYYYMMDD_experimentname)
?. [Prepare software]
Save sample (adults/L4s etc)
?. [Prepare software]
CompartmentPressure (psi)Sheath~2.6Sample cup~1.6Diverter6Cleaning4
CompartmentPressure (psi)Sheath~2.6Sample cup~1.6Diverter6Cleaning4
?. [Clean system]
Maintenance -> Flush Sample
?. [Clean system]
Click ‘Refill Sample’ – the sample cup pressure should decrease. You can see this in the software on the left hand size (include screenshot).
Sometimes the sample cup pressure doesn’t decrease and in fact increases. You can still unscrew the sample cup but if this persists there may be a blockage.
?. [Clean system]
Unscrew sample cup and replace with 50ml falcon filled with 50ml 20% Decon-90 (D901-04; dilute in sterile water)
?. [Clean system]
Once securely replaced click ‘Done refill’
?. [Clean system]
Check ‘Sample on’ and ‘mixer on’ – Decon solution should now pass through the system; allow a 50 ml to pass through (make sure sheath is unchecked)
You will get a warning about contaminating the flow cell, this normal and you can click 'Yes'
?. [Clean system]
Uncheck ‘Sample on’ or click Abort to stop sample flow.
?. [Clean system]
Repeat steps 13-17 with cleaning solution (Product Number: 300-5072-000; pink in colour) but only pass 10ml through the system
?. [Clean system]
Repeat steps 13-17 with a falcon filled with 50ml sterile water (from 4thfloor kitchen) and pass >25ml through the system
?. [Flow check]
Hold an empty 50ml faclon under the sample dispenser (otherwise you will end up with a puddle of water on the COPAS!)
?. [Flow check]
Maintenance -> Flow check
?. [Flow check]
Allow the water to flow into the falcon tube for the full 60 seconds
?. [Flow check]
Record volume in the COPAS logsheet (Volumes/behavgenom$/Documentation/Protocols/COPAS_wormsorter/COPAS_logs/COPAS_logbook.xlsx)a.Aim for flow of 25ml/minute
?. [Control particles]
Prepare 8mL solution of 1X control particles (Product number: 310-5071-001; 2 mL 4X stock solution, 6mL sterile water; label tube with date and wrap in foil to keep in the dark) – to be prepared once per week
?. [Control particles]
Set up -> Control Particles
?. [Control particles]
Acquire
?. [Control particles]
Adjust Sample Cup pressure so that you have 10-20 events/sec
?. [Control particles]
Aim for TOF Cv around 10-15% and tight narrow peak for TOF and Extinction
?. [Control particles]
Record TOF and Extinction mean and cv in Volumes/behavgenom$/Documentation/Protocols/COPAS_wormsorter/COPAS_logs/COPAS_logbook.xlsx
?. [Control particles]
Take a screenshot of the control particles screen and save into Documents/BehaviouralGenomics/ControlParticlesa.Print Screenb.Open Paintc.Ctr + vd.Save
?. [Control particles]
Set up -> control particles to exit
?. [Control particles]
Refill sample with sterile water, run 10 mL water. You are now ready to load your sample
?. [Load sample]
Refill sample
?. [Load sample]
Gently swirl falcon with worms and screw into sample cup
?. [Load sample]
Done refill
?. [Load sample]
Turn mixer ON. If you do not do this you may lose all your worms that have settled to the bottom of the tube!!!
?. [Load sample]
Maintenance -> Prime Flow Cell; to flush sample through the system and remove air bubbles
?. [Load sample]
Maintenance -> Flush sample
?. [Load sample]
Check 488nm (and 568nm) laser boxes
?. [Load sample]
Check ‘Use sort gate’ for stored sort gate
?. [Load sample]
Click ‘Acquire’ – sample should pass through the system and number of events per second will be shown:a.Aim for 5-20 events per secondb.If too few/too many events increase/decrease ‘Sample cup pressure’ so that it is between 1.5-2psic.Gate recovery should be between 20-50% d.To ensure only one event per droplet go to Setup->Coincidence, select ‘Pure, no double’. This increases accuracy in the number of worms dispensed but the time to dispense may increase.
?. [Test dispensing]
Click on the plate icon on the top bar
?. [Test dispensing]
Select number of objects to sort
?. [Test dispensing]
Select the wells you would like to fill (for testing we use a spare 60mm plate and fill wells A1, A2, B1, B2)
?. [Test dispensing]
Select which gate to use (R4)
?. [Test dispensing]
Apply
?. [Test dispensing]
Place 60mm plate in front left corner of left-hand stage with A1 in the left corner.
?. [Test dispensing]
Click ‘Fill plate’ (the sheath will automatically turn on)
?. [Test dispensing]
Keep an eye on the number of events per second
?. [Test dispensing]
Ensure the ‘Diverter pressure’ is checked
?. [Test dispensing]
Check under microscope that the correct number of objects were dispensed per ‘well’
?. [Test dispensing]
If too many objects, decrease sample cup pressure and repeat steps 8-11 or select Pure no double to increase accuracy.
?. [Fill plate]
Click on the plate icon on the top bar
?. [Fill plate]
‘Clear plate’
?. [Fill plate]
Select number of objects per well and click ‘Apply to All’ or select which wells you would like to fill.
?. [Fill plate]
Apply
?. [Fill plate]
Place 96 well plate in left-hand stage (A1 aligned)
?. [Fill plate]
Ensure ‘Diverter pressure’ is checked’; if it is not then liquid comes out of the dispenser constantly and you get flooding.
?. [Fill plate]
‘Fill plate’
?. [Fill plate]
Each time you do a dispense, save the file in the convention: YYYYMMDD_strain_ _platenumber in Documents/yourname/experimentname/date (eg. 20210211_PHX1675_S01R01)
?. [Fill plate]
Keep an eye on the number of events per second still and monitor how much sample fluid is coming through the system
?. [Clean system]
Repeat steps 13-19
?. [Clean system]
Keep sample cup with 50ml water in the falcon secured so that the system is air-tight and closed
?. [Clean system]
Empty recovery cup and waste bottle on floor
?. [Clean system]
Remove waste tray (see below), unscrew, and empty and spray with water
?. [Clean system]
Turn off all equipment in this order:a.Softwareb.Sorterc.Pumpd.Computerse.Lasers