Prepare your samples by measuring their PCV (or estimating the cell amount according to the doubling rate). Dilute them with PBS in order to have between 200 and 500 cells/µl. Prepare at least one well that has seed cells.

Steps 1 to 4 are optional and should be done from time to time.

1. Trash the waste on the bottom right of the machine if it is full before you start.

2. Put tubes with bleach (detergent) at the right positions.

3. Run 'Cytosoft 5.3'

4. Click Clean and Shut Down -> This would take around 15 min.

5. After cleaning, click Guava Express Plus on the left column.

6. Go to Analysis mode and click 'Open Data Set'.

7. Go to the 'iGEM' folder and open the 'Setting' file.

8. Go to Acquisition - and hold here.

9. Go to the desktop and run WorkEdit 5.3.

10. Highlight the wells you are going to use, check "Acquire this sample".

11. Label them as Guava Express Plus, check the "Mix for 3 seconds", set the speed from high to medium. Optionally, fill the sample ID and the dilution factor.

12. Save and go back to Cytosoft 5.3.

13. Go to Acquisition, start the worklist.

14. Place the 96-well plate into the tray, make sure the A1 well is where it should be.

15. Name the file as 'Today's date_title'

16. When you are asked to adjust the settings, check a well that contains seed cells.

17. Compare with the worklist, check if the flow and the amount of cells detected are reasonable.

18. Click "Next step" and then "Resume".

19. Wait until all the wells are measured - data will be saved automatically.

20. Take the 96-well plate out and insert the tubes that are required for cleaning.

21. Go to Main menu and click 'Clean and shut down'.