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Utilizing FlowJo for Compensation Calculation in Multi-Color Flow Cytometry

This guide details the process of using FlowJo to calculate compensation for multi-color flow cytometry data, specifically on a Calibur. It includes steps for organizing files into groups, accessing compensation controls, and generating positive and negative populations from single stain histograms. Key features include utilizing the histogram bisector tool for clear separation of populations and defining compensation matrices in FlowJo. The final step covers how to identify compensated parameters visually, ensuring proper data interpretation.

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Utilizing FlowJo for Compensation Calculation in Multi-Color Flow Cytometry

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  1. Using FlowJo to calculate compensation 060502 Emory Vaccine Center FCC / Suzanne Mertens / saguila@emory.edu

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

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

  4. 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

  5. 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

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

  7. 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.

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

  9. Last step - select to use the new matrix

  10. 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

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

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