Calibrated Parameters: Overview
FlowJo has a unique platform that allows you to calibrate any collected parameter. This gives you the ability to express any parameter's values (graphical display, any statistical calculations) in the units of your choice. Most commonly, this is used to convert the scaling into absolute number of molecules (given a standard that converts between the fluorescence intensity collected on your instrument and absolute numbers of fluorophores).
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.
Callibrate your samples to a reference when you have collected data on a standard sample which you can use to convert one unit of measure to another. (for example, a commercial bead set where each bead has a defined absolute intensity mapped to a known unit value; 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 saved with the workspace in which they were created permanently. They are accessible as Derived parameters; thus, if you want to apply a Calibration Standard to another sample (after defining it), select Windows > Open Compensation/Derived Parameter Editor select its name in the Derived Parameters list click the Assign Tubes tab and drag tubes in from the Workspace. For more information on derived parameters, click here.
When you select Calibration under the Tools menu, you will be presented with the Define New Calibration Standard dialog window. It asks you to define which parameter you want to calibrate. For detailed instructions on creating a new Calibration Standard, click on this link.
Download a Calibration Workspace with Demo Data to try out this platform.
