Ozette Resolve
Spectral Unmixing

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Unmixing Single Stain Controls

Click Unmix 

Once you have created your control set, executed all gating, and made any updates to the Source of Background, you may click “Unmix” at the bottom right of the page.

This will trigger the unmixing algorithm to unmix the single stain controls.

Note that the unstained and assay control files will NOT be unmixed.

This can take time depending on the number of fluors in your panel and the number of events in each control.

Click “View Results” to view the status of the unmixing run and the unmixed results.

Unmixed Single Stain Controls

Once unmixing of the controls is complete, the View Results page will populate with metadata, downloadables, and visuals that can be used to QC the results.

  1. Downloadables:
    • Cofactor file: This file contains estimated transformation parameters for each fluorochrome in the panel. For downstream analysis, we recommend that each fluor in output Resolve data be transformed by the hyperbolic arcsine function with the cofactor value listed in this file.
    • Bulk export: All unmixed control files
    • Individual files: Single control downloads
  2. Nx1 Plots:
    • Click on individual file names on the View Results page to view the Nx1 plots for that file.
      • These Nx1 plots show unmixing of the root population. These visuals can be used for basic QC review of the results, but do keep in mind that these plots include dead cells and debris that may be gated out in your final downstream analysis.
    • You can toggle between controls using the arrows at the top left of the page.
    • By default the assigned fluorophore will be selected but you can change that using the “X” dropdown menu.
    • You can change the axes settings between the range of the data and the range of the instrument using the “Display Range” menu.

If you identify any suboptimal results in the unmixed  controls, you may iterate on your control sets using the Duplicate and Inheritance features described above. Continue iterating on the control set and unmixing your  controls until you are satisfied with the results.