How can I recreate ratiometric data acquired in FACSDiva?

It is possible to acquire data with BD FACS Diva ratiometrically, meaning one parameter is divided by another such as FL1/FL2, to create a new parameter representing these values. The resulting ratio parameter will be stored as a new parameter with the .FCS file and this parameter is dynamically calculated in BD FACSDiva through the use of a special keyword that is stored in the header.

To display the ratio in FCS Express, you must complete a few simple steps to construct a parameter math formula to either use the FACS Diva method for creating the parameter or the FCS Express method. The benefit of using the FCS Express method to calculate the ratiometric data is, that unlike BD FACSDiva, the new parameter can use the actual ratio value rather than a value created by using a multiplier. If you would like to create a new ratio parameter using the FCS Express method, please see the Using Parameter Math topic for details on creating a simple ratio parameter.

Recreating a ratio parameter from BD FACS Diva data

1. Click on the Tools tab–>Transformations->Transformations.

2. In the Transformations navigator, click the down arrow to the right of the blue plus->select Parameter Math.

3. Once the Template File and Gate, if needed, have been selected, click New to access Create New Sequence Item dialog.

4. Click on the Formula radio button in Create New Sequence Item dialog.

5. Enter the following formula, where X is the parameter number of the numerator parameter and Y is the parameter number of the denominator parameter:

=(pX/pY)*

6. Right click next to * symbol.

7. Choose Insert Token from the drop down menu.

8. Choose Keyword in the Insert a Token dialog

9. Choose the Keyword category.

10. Enter the keyword PXSF, where X is the parameter number of the ratio parameter stored by BD FACS Diva. In the example below the keyword parameter number was 6.

11. Click OK.

12. Drag and drop newly created parameter math from Transformations navigator to intended plot.

You can now click on the axis of your plot to access the newly created ratio parameter, which will look as it did in BD FACSDiva. FCS Express will use the compensation, if any, applied to the plot, and “Parameter Math applied” will be appended to plot title.