There are flow cytometry platforms that do not export Time as a parameter for their data.

In FCS Express, you can create a Time parameter for data files that do not have a Time parameter that was exported with the file.

To do this, we will use 3 features:

• Keyword Tokens
• Custom Tokens
• Parameter Math

To summarize, Parameter Math will allow us to create new parameters for our data by using existing features of the data.  In this example, we will:

• use a keyword token to extract the total number of events within the file,
• create a custom token to help us determine the amount of time it took to acquire the data, by using the keywords $BTIM and $ETIM in a function, MinutesBetween, that extracts the amount of minutes between each time point.

Please note that if your data lacks a Time parameter, the data file will lack the information to provide FCS Express with the arrival times for each event.  As a result, FCS Express will equally space each event between the begin time and the end time information stored within the data file.

1. To get started, open a new blank layout by going to File > New > New.
2. Insert a plot that contains your Time-less data file.
3. From the View tab, open the Custom Tokens window.
4. Add a new Custom Token by clicking on the + sign in the Custom Tokens toolbar.
5. Rename the custom token Minutes Between.
6. For the custom token formula, add an = sign, then right click to select the Insert Function menu.
7. From the Date-Time category, select the function MinutesBetween(start time, end time).   *If you would like to view Time in a unit other than Minutes, you can use other Date-Time Functions, such as HoursBetween, SecondsBetween or MillisecondsBetween.
8. Within the function, we will need to replace start time and end time with the actual time points coming from our data.  We can use the keywords $BTIM and $ETIM for this.
9. Clear out the information between the parenthesis and then right click within the parenthesis to select Insert Token.
10. Select Keyword from the Insert a Token window and then click Insert.
11. In the Create Keyword window, select the Keyword category and then either type the keyword $BTIM in the field or click on the ellipsis to browse for the keyword within the data file., then click OK.
12. Add a comma after the keyword that we just added and insert another keyword token, this time for the keyword $ETIM.  Close the parenthesis at the end of the function.
13. At this point, your function should now look like this.
14. Close the Custom Tokens window.
15. From the Tools tab, open the Transformations window.
16. Within the Transformations window toolbar, click on the triangle next to the + sign.
17. Select Parameter Math and rename the transformation Calculated Time.
18. Under the Transformation Options section, click New.
19. Within the Create New Sequence Item window, enter Calculated Time for the Output Parameter Name.
20. Select the Formula option.
21. Within the Formula field, enter (row-1)*(.
22. After the open parenthesis, right click and select Insert Token.
23. From Custom, select the Minutes Between custom token that we created in Step 5.
24. After the token, add a /, then right click and select Insert Token.
25. Select Keyword, then in the Keyword category, type in $TOT for the keyword to use, then click OK.  This is the keyword for the total number of events within the data file.
26. After the $TOT keyword, add a ) to close the formula and then click OK.
27. At this point, you should see something similar to this:
28. To apply the Parameter Math to your data, drag the transformation onto a plot.
29. The title of your plot should now reflect that Parameter Math has been applied.  The new parameter can now be selected from the X or Y axis.