Using FlowJo to calculate compensation

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Using FlowJo to calculate compensation

• 060502
• Emory Vaccine Center
• FCC / Suzanne Mertens / saguila_at_emory.edu

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FlowJo can be used to calculate compensation for
multi-color data acquired on a Calibur
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Organize your files into groups

• First place your comp tubes into a separate
  group
• The put your experimental tubes into a separate
  group

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Accessing FlowJos 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

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Generate positive and negative populations from
single stain histograms
In this example, the FITC single stain has been
gated for lymphocytes, then displayed as a FITC
histogram
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Click and use the histogram bisector tool to
quickly separate positive from negative
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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.
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Compute and save your compensation matrix

• Click Compute
• Enter a name for your matrix

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Last step - select to use the new matrix
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FlowJos indicator for compensated samples is
easy to overlook

• The purple vertical bar is your indicator that
  compensation has been applied to these samples

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