Once you have created a calibrated parameter, it is added to your sample as if it were just another parameter. You can apply the calibration standard to any other sample in your collection (and you can apply the calibration to an entire group, if you wish). You may have as many different calibration standards in a workspace as you want; in fact, you can even apply multiple calibration standards to the same sample (a new parameter will be added for each calibration that you apply).

When you graph your data, select the calibrated parameter on the axis of choice--the scale values on the axis are now drawn in your calibrated units! For example, if you calibrate to absolute number of molecules per cell, then you can display these numbers on the axis. In addition, any statistics you calculate (medians, CVs, etc.) will be expressed in units of absolute molecules. Finally, you can even enter a gate based on those units: you could, for example, specify a gate on all cells with more than 5,000 molecules of an antigen.

There are two methods by which you can calibrate a parameter: manual or reference. Use the manual mode when you want to enter the exact conversion values yourself. Use the reference when you have collected a standard which you can use to convert to your units (for example, a commercial bead set where each bead has a defined absolute intensity; or, a sample stained with an antibody that has a known binding capacity to the cells). When you use a reference, FlowJo automatically finds all of the peaks in the fluorescence, and asks you to enter any or all of the calibration values for those peaks. FlowJo then performs a linear (or logarithmic) least squares regression to determine the best calibration value, and creates the calibrated parameter.

A "Calibration standard" is FlowJo's internal representation of the conversion of fluorescence to absolute units. Calibration standards are stored in the workspace, permanently. They are accessible as Derived parameters; thus, if you want to apply a Calibration Standard to another sample (after defining it), select the appropriate derived parameter under the "Derived Parameters" menu item. For more information on derived parameters, click here.

To define a calibration standard, you will be presented with the "Define New Calibration Standard" dialog window" that asks you to define which parameter you want to calibrate, and then which method to use. This dialog window is shown when you select "Calibrate..." under the Workspace menu. To see how to use either method to create a new Calibration Standard, click on the link to this dialog window.

In addition, you can see two examples of creating Calibration Standards. Both use reference samples; one uses a bead set, the other, the expression of CD4 on lymphocytes as a defined set point. Examine these two series of pages to see how easy it is to calibrate your samples with FlowJo!

Download a Calibration Workspace with Demo Data to try out this platform.