Using FlowJo to calculate compensation Emory Vaccine Center FCC / Suzanne Mertens /

FlowJo can be used to calculate compensation for multi-color data acquired on a Calibur

Organize your files into groups First place your “comp” tubes into a separate group The put your experimental tubes into a separate group

Accessing FlowJo’s compensation controls FlowJo expects to see defined positive and negative populations for each single stain The user must go ahead and do proper gating to create single stain histograms with positive and negative populations

In this example, the FITC single stain has been gated for lymphocytes, then displayed as a FITC histogram Generate positive and negative populations from single stain histograms

Click and use the histogram bisector tool to quickly separate positive from negative

Drag and drop gated populations to define the matrix Drag your gated populations to the “AutoAssign Box” amd FlowJo will sort out which population goes in the right Definitions box.

Compute and save your compensation matrix Click “Compute” Enter a name for your matrix

Last step - select to use the new matrix

FlowJo’s indicator for compensated samples is easy to overlook The purple vertical bar is your indicator that compensation has been applied to these samples

Remember to view compensated parameters Uncompensated parameters have no brackets Compensated parameter have brackets