![]() ![]() There are some useful principles to follow: However, due to subtle differences between instruments, we always recommend reviewing the manufacturer’s instructions. The procedure for setting the correct fluorescence compensation follows the same basic principles on any cytometer. Hints and tips for applying compensation in multicolor flow cytometry Compensation removes false positive signals caused by spectral overlap. If you have any questions about spectral compensation we’re here to help, send some examples to for a closer look.Figure 5. Once spectral compensation has been performed, the results can be previewed and applied to samples in much the same way standard compensation is treated. Note: Because of the immense amount of computation required for this optimization feature, it can take some time to complete, so be ready for a coffee break. Running the Optimize Weights function for spectral compensation will utilize currently selected compensation control populations to compare hypothetical spillover spreading matrices (SSM) for different weights and adjust those weights to minimize the SSM. Samples used for primary detectors also help to name those parameters after spectral compensation. Once that assignment is confirmed and/or adjusted as necessary, you can click in the sample column to the left of an unused detector and choose “Remove Unused Parameters” to quickly trim away non-primary channels. FlowJo will try to automatically assign the proper control sample to its respective detector. When first running spectral compensation, researchers are prompted to choose their primary detectors – We generally recommend selecting all detectors to start with. The primary control samples are used for parameter naming. Using spectral compensation means that signal from non-primary detectors are still used to enhance the true signal of markers where possible.
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