Enter your fluorescence channel names and spillover percentages into the Flow Cytometry Compensation Calculator to get your Compensation Quality Score, Total Spillover, Maximum Correction Factor, and Matrix Determinant for assessing spectral overlap.
Compensation Quality Score
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Total Spillover
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Maximum Correction Factor
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Matrix Determinant
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Compensation is the mathematical correction for fluorescence spillover between channels in multi-color flow cytometry. It removes unwanted signals from overlapping fluorophore emissions to improve data accuracy.
Use single-stained controls for each fluorophore in your panel. Measure the median fluorescence intensity in each channel and calculate the percentage of signal appearing in non-target channels.
A compensation quality score above 85% indicates excellent compensation. Scores between 70-85% are acceptable, while scores below 70% suggest you may need to optimize your panel or spillover measurements.
The matrix determinant indicates the mathematical stability of your compensation matrix. Values close to zero suggest the matrix may be poorly conditioned, leading to amplified noise in compensated data.
This calculator is designed for conventional flow cytometry. Spectral flow cytometry uses different unmixing algorithms and typically requires specialized software for optimal compensation.
Recalculate compensation whenever you change fluorophores, antibody lots, instrument settings, or if you notice poor separation in your data. Many labs recalculate monthly or with each new experiment batch.
High spillover typically results from overlapping emission spectra of fluorophores, suboptimal filter combinations, or using fluorophores with broad emission profiles in adjacent channels.