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Using FlowJo to calculate compensation 060502 Emory Vaccine Center FCC / Suzanne Mertens /...
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Transcript of Using FlowJo to calculate compensation 060502 Emory Vaccine Center FCC / Suzanne Mertens /...
Using FlowJo to calculate compensation
060502Emory Vaccine Center
FCC / Suzanne Mertens / [email protected]
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