TITLE: COPAS wormsorter
AUTHORS: 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 the compressor at the wall – it should show a pressure of 40psi after switched on
?. [Prepare equipment]
Turn on COPAS machine with switch on the left hand side
?. [Prepare equipment]
Turn on the lasers (488 laser sufficient if using unmarked animals). Add in picture of lasers.
?. [Prepare equipment]
Turn on the computer
?. [Prepare equipment]
Discard waste contents that are in the recovery cup (small shallow cup on the left-hand side of the machine)
?. [Prepare equipment]
Check that there is water in the sheath. If the water is low, fill up with MQH2O (not M9).
?. [Prepare equipment]
Make sure that the recovery cup and sample cup are securely tightened so that there are no leaks in the system
?. [Prepare software]
Open dbgview – should always be running in the background
?. [Prepare software]
Open FlowPilot software and a prepared experiment with a set gate for eg Adults. :
?. [Prepare software]
File -> Load Experiment
?. [Prepare software]
File -> Load sample
?. [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 falcon filled with cleaning solution (pink in colour)
?. [Clean system]
Once securely replaced click ‘Done refill’
?. [Clean system]
Check ‘Sample on’ and ‘mixer on’ – cleaning solution should now pass through the system; allow a 2-3 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 11-15 with water
?. [Load sample]
Repeat steps 11-13 with sample.
?. [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 – include screenshot of software here
?. [Load sample]
Click ‘Acquire’ – sample should pass through the system and number of events per second will be shown:Aim for 10-20 events per secondIf too few/too many events increase/decrease ‘Sample cup pressure’ so that it is between 1.5-2psiTo 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
?. [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’
?. [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
?. [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]
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 11-15
?. [Clean system]
Keep sample cup with water secured so that the system is air-tight and closed
?. [Clean system]
Turn off all equipment (Computer, lasers, compressor, worm sorter).