1. IF no compensation is necessary, Select
Detector and Threshold Settings.
2. Adjust FSC gain and BSC gain such that your
cells are on scale.
Acquire enough events for the sample to be
sorted so that you can set gates. Pause the
Create plots on the worksheet. Double click
inside gates to create gated plots, or change
the gates by clicking on the gate name at the
top of a plot. Be sure to include a singlet
For experimental sample gain settings
Adjust gate settings for populations of
i. Set the value to record to 5,000 or 10,000
events. If your positive population is rare,
record more events.
5. Sorting into Tubes
1. The Sony pre-calculates the sort stream voltage and angle automatically. If you have large cells, or want to test the
sort stream location before sorting:
i. Open "settings" option in the cytometer ribbon
ii. Open "advanced settings"
iii. Load test sort tubes into the collection device
iv. Click "load collection." Sort streams should come up with assigned voltages.
Test sort only works if sample is NOT loaded
vi. Quickly turn on and off the sort test and verify that the assigned voltages produce streams that will go inside
2. Continue your sort setup: Select the desired sort mode. Purity or Semi-Purity is recommended. (f)
3. Set the L and R sort gates. Indicate the number of
cells to be sorted, or leave the value at 0 to sort
4. Place the collection tube holder into the collection
area, and set collection tubes.
5. Click 'Load Collection' (d)
6. Make sure flow rate is stable (a), click 'Sort Start'
7. Press 'Record' to save data for the sample. (c )
8. Run the sample pressure at a maximum of 6
9. From the Cytometer tab, you can adjust
temperature control for the sample and collection,
chamber lights and agitation.
10. Monitor the collection tubes, and change them
when they fill.
i. To change tubes: Pause the sample, Unload
Collection, switch tubes, load collection and the
sort will continue.