Styx

Styx is used to sort droplets based on fluorescence.

Procedure

Good practice to check under the microscope first to see if there is any fluorescence.

1. Arrangement:
   1. Spacing oil – usually a large quantity (e.g., in a 5 mL Eppendorf or a 25 mL tube), tubing need not do all the way down to the bottom.
   2. Sample – insert the tubing all the way down and as you keep screwing gradually pull it out such that it's at the very bottom but not touching the tube.
   3. Empty
   4. Waste – no pressure control or screws needed
   5. Positive selection – no pressure control or screws needed
2. Purging – use Start button:
   1. Spacing oil: 3 kPa – wait until it fills the chip
   2. Sample: 1 kPa
      1. Do not plug into the chip yet.
      2. Wait for the spacing oil to fill in while holding the tubing upwards (so that the liquid doesn't go out).
      3. Once the oil is in, lower the tubing into a clean Eppendorf and wait until excess oil is fully drained an a couple of drops with droplets accumulate at the tip of the tubing.
      4. Plug into the chip.
3. Running:
   1. Spacing oil: 12 kPa (or 21 kPa)
   2. Sample: 8 kPa (or 15 kPa)
   3. Voltage: 700-800 V – for best quality, make sure the sorted droplet goes into its outlet without touching anything; can also adjust pulse duration to 3000 us and pulse delay to 0 us
   4. Laser: align to the right tip of the electrode
   5. Start the laser(s) of the desired wavelength
   6. Start fluorescence data collect (in the fluorescence tab)
   7. Start voltage
4. Clean up:
   1. Take out the tubing of positive and waste and hold vertically for the droplets to drain into the tubes
   2. Stop the experiment (voltage, laser, and fluorescence detector)
   3. Disconnect everything