FACS general protocol

This is a brief protocol for using a cell sorter. Check with the responsible of the equipment in case there could be some variations in your workplace.

Setting up and sorting

Step	Explanation
1. Turn on the equipment	Use power button on the left side
2. Verify that all tanks were properly treated by the last user	Check that flow buffer and ethanol tanks are filled and waste tank empty
3. Connect tubing for operation	Air tubing is transparent (mounted on top), flow tubing is blue and mounted below the filter
4. Turn on computer and open software	Use login provided by the center
5. Check flow cell and sorting chamber	Clean them if dirty
6. Check that closed-loop nozzle is inserted in flow chamber	If not, insert it
7. Run fluidic system startup	Follow software instructions
8. Clean selected nozzle and replace it in the flow chamber	Use ethanol
9. Initiate fluid stream checking that enters the waste	Follow software instructions, fluid stream should not change
10. Wait ca. 30 min	To stabilize the fluid stream
11. Check if stream is stable and set the gap	Virtual drops should be visible in the top of the screen, break-off should be visible, drops should be symmetric, satellite drops should merge with drop as fast as possible (not more than 5 satellites).
12. Check the performance using the software	Follow the software instructions
13. Create a new experiment	Follow the software instructions
14. Create New Sorting Template or import sort template	Follow the software instructions
15. Toggle Sweet Spot ON	Follow the software instructions
16. Check that sorting streams enter collection tubes	Load tubes in collection holder, turn on voltage, open laser cover, close waste drawer, press test sort. If sorting streams do not enter collection tubes, direct it via slides and voltage in sorting window
17. Open sort layout	Choose the appropriate sorting mask
18. Sort test	Use template beads to test the sorting
19. Set the drop delay	Follow the software instructions
20. Perform the sorting	Load the sample into the loading chamber

Cleaning and shutting down

Step	Explanation
1. Clean the sample line	Follow the software instructions. Use bleach, detergent and distilled water in this order
2. Clean the nozzle	Turn off the stream, take out the nozzle from the flow chamber and clean it distilled water and ethanol
3. Clean the flow chamber	Connect the closed-loop nozzle and use distilled water at maximum rate
4. Prepare fluids shutdown	Follow the software instructions
5. Vent the air pressure from the flow buffer tank	Pull up the ring on the pressure valve on top of the FACS flow tank
6. Turn off flow cytometer and computer	Use button on the left side of the equipment
7. Prepare tanks for the next user	Refill flow buffer tank and empty the waste tank