In this case, only a single reference value needs to be entered: 50,000 (molecules) corresponding to peak number 3. FlowJo will assume that the calibrated parameter has the same dynamic range (same number of decades) as the original parameter. In the absence of a much better calibration standard, like a multi-bead set, this assumption must be made.

Once you are satisfied, you can click on "Define Parameter"; in the dialog window (below), the name for this standard is "Absolute Molecules".

At this point, the sample has the "Absolute Molecules" parameter, corresponding to absolute numbers of molecules, already added. If you open the lymphocyte gate and display a histogram of this parameter, you will see the graph:

Note that the X-axis scaling is such that the CD4 peak is now right at 50,000. Here, you could choose to manually define a gate (Select "Manually enter gate" under the Graph menu) to gate on cells which have, for example, more than 10,000 molecules of CD4.

At this point, you would like to apply this calibration standard to another sample. Select the other sample, and from the "Derived Parameters..." menu, select the submenu that corresponds to your new standard:

Now the other sample has this parameter as well. In our case, we applied the absolute scaling to a sample stained with CD8. At this point, gates were defined on CD4 and CD8 positive cells in the two samples, and the median fluorescence was calculated for all populations. The workspace is shown below:

In this workspace, you can see that CD4 T cells have 50,185 molecules per cell. The "Others" gate was for negative (autofluorescent) cells; in this experiment, autofluorescence of lymphocyte corresponds to less than 200 molecules. The CD8 stain shows up with 66,107 molecules. This is expected, since CD8 is expressed at higher levels than CD4. Again, the negative cells show up with less than 200 molecules of background fluorescence.

Go to the overview of Calibrated Parameters.