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.