You can open the Calibration platform three different ways:
- Select a sample or gated population in the Workspace and then choose Calibration from the Tools menu.
- Right-click on a sample or gated population and select Calibration...
- Add the Calibration button to the Workspace, select a sample or gated population and press the Calibration button.
FlowJo will open the Define New Calibration Standard window, as shown below.
Next, choose from the X-axis the parameter you want to calibrate. By default, FlowJo searches for the parameter that most likely corresponds to phycoerythrin staining (PE), since this is the most commonly calibrated parameter. In the example below, APC is shown.
FlowJo will identify the reference peaks, number them and provide the Scale values for those peaks. Then, add the reference values (molecules, MESF/ABC, etc.) associated with each peak in fields of the Absolute Units column (shown below). With each addition, FlowJo will calculate a least squares regression (linear or logarithmic) along with the correlation coefficient.
Mouse over the red dots for more information
Download a Calibration Workspace with Demo Data to try out this platform.
Go to the overview of Calibrated Parameters.
For this example, an 8-bead set was collected; singlets were gated by Forward and Side Scatter. FlowJo identified 8 peaks in this histogram. Here you are asked to enter the absolute number given for a calibration product for one or more of these peaks. As you enter values, FlowJo continually performs regressions and graphs the data points and the regression overlayed on the histogram. The final scaling of the calibrated parameter will be shown as a vertical axis on the histogram. Select the parameter that you would like to calibrate. As reference values are added to the Absolute Units column, your slope and correlation coefficient will appear here. If you have a slope at or near one, you may want to double check your reference values. Once you are satisfied with the fit, click on Define Parameter.
