2. Check to be sure the buffer containers are full and the waste tank is empty.
3. Select Startup and the instrument will flush the system and load sheath in ~12min.
4. Press Load and load two drops of FlowSight Instrument calibration beads. Vortex first.
5. In the ASSIST view, press Start all calibrations and tests. Calibration takes ~12 minutes and when all tests pass the
system is ready to run for the day.
6. Select load default template or experiment template from the File menu.
7. Press Load and load an aliquot of a sample with each fluorochrome present. Run on 'Low' speed for best sensitivity.
8. In the Illumination section, turn on the appropriate lasers for each fluorochrome in the experiment.
9. Adjust the laser power to maximize brightness and prevent saturation. The 405 laser power should be adjusted to
use a minimal acceptable power to prevent autofluorescence and also PE excitation into the BF9 reference channel.
10. Create dot plots and regions to identify the cells to collect, or collect 'All' events.
11. Set the acquisition parameters including file name, destination folder, number of events and population(s) to col-
lect.
12. Choose file format, either .rif (IDEAS), .fcs (FACS software) or both from File menu.
13. Compensate data if needed. Data can be recompensed in IDEAS
®
Software post acquisition.
a. Open the compensation wizard.
b. Load a compensation single color sample.
c. Verify the system finds the correct channel.
d. Set the population to save.
e. Set the acquisition file name and destination.
f. Press next to save the data file.
g. Repeat steps B through F for each compensation sample. Press exit and save to return to the experiment.
14. Continue collecting all experiment files using consistent instrument settings (In general, brightfield will be in chan-
nels 1 and 9, SSC ~40 mW in channel 6; and the cells to collect R1 using brightfield area vs. aspect ratio to identify
single cells).
15. Save an experiment template by selecting Save Template from the File menu.
For Research Use Only. Not for use in diagnostic procedures.
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Amnis
®
FlowSight
®
Imaging Flow Cytometer User Manual