Understanding and Perfroming Multiple Color Compensation on the Flow Cytometer
Flow cytometers use dichrioic mirrors and band pass filters to discriminate the different colors employed. Because each fluorochrome has its own unique 'signature' or emission spectrum often the leading or trailing end of one color can have wavelengths that enter into another filter set causing unwanted signals. To get around this a compensation network is utilized to subtract any undesirable signals. It is important to set up your experiment with single color samples to perform this compensation. An example and data analysis on normal mouse thymocytes with what to expect as one attains the proper compensation is provided below. The CD4 and CD8 are directly conjugated antibodies. The CD3 is biotin conjugated and a second (indirect) labeling with Streptavidin APC is employed.
Sample Tube #1 HBSS
No compensation applied.
Sample Tube #2 CD4-PE
No compensation applied.
Sample Tube #2 CD4-PE
Partial compensation applied.
In this example 0.4% PE is subtracted from the FITC PMT. (PMT2-0.4% PMT3) and3.3% PE is subtracted from the APC PMT. (PMT5-3.3% PMT3).
Sample Tube #2 CD4-PE
Correct compensation applied.
In this example 1.4% PE is subtracted from the FITC PMT. (PMT2-1.4% PMT3), and 6.5% PE is subtracted from the APC PMT. (PMT5-6.5% PMT3).
Sample Tube #3 CD8-FITC
No compensation applied.
Sample Tube #3 CD8-FITC
Partial compensation applied.
In this example 9.0% FITC is subtracted from the PE PMT. (PMT3-9.0% PMT2).
Sample Tube #3 CD8-FITC
Correct compensation applied.
In this example 12.5% FITC is subtracted from the PE PMT. (PMT3-12.5% PMT2.
Sample Tube #4 CD4/8/SA-APC
Sample Tube #5 CD4/8/3-B & SA-APC
Mario Roederer, Ph.D. Comepnsation Tutorial
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Last updated: 7-December-04
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