User's Manual

27
Preparation of droplets for flow cytometry
1. Remove tubes with dPCR droplets from the PCR machine.
2. Make sure the 2x dPCR buffer ● is diluted with molecular grade water to 1x. Mix well by vortexing
for 10 seconds or inverting the tube at least 20 times.
3. Stain droplets with Droplet dye ● as follows:
• Prepare flow cytometry buffer by adding 1ml 1x dPCR buffer to a flow cytometry tube (tubes
depend on flow cytometer instrument).
• Spin down Droplet dye at 5000 rpm ● 2 minutes.
• Add 10 μl Droplet dye ● into the flow cytometry tube with 1ml dPCR buffer. Mix gently to
dissolve the dye in the dPCR-buffer.
• Remove the supernatant from the PCR tubes containing the dPCR droplets leaving the droplets
undisturbed at the bottom.
• Use 200 μl buffer from flow cytometry tube to transfer all droplets from the PCR tubes to the
flow cytometry tube. Use tips that minimize binding of droplets to the side of the tip.
• Leave at room temperature protected from light for 5 min to stain droplets.
4. Add 15 μl of molecular grade H
2
O into the bottom of a 1,5 ml DNA LoBind collection tube and place
the collection tube in the appropriate holder in the cell sorter instrument.
5. Confirm that the sort settings for your cell sorter are correct. Check that the side stream is
centered on the collection tube.
6. Load tube containing the stained dPCR droplets in buffer on the flow cytometer and start analysing.