Ozette Resolve
Spectral Unmixing

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Appendices

Appendix A: File Format Requirements

  • Control Files: .fcs format from supported instruments
  • Metadata .csv files: One file per row, headers should be exactly as specified in the downloadable templates
  • Fluorophore .txt file: One fluorophore per row, no headers

Appendix B: Generating Good Controls for Resolve

Understanding the composition of your controls is an important step in the Resolve workflow. This is because Resolve uses the measurements in the unstained control files to estimate the autofluorescence contribution in individual events, and uses the measurements in the single-stain controls to estimate event-specific spectra. For these estimates to be optimal, you should make sure that you include in your controls events of all types/lineages you plan to measure in the full-stain files, while eliminating non-specific and irrelevant sources of fluorescence.

For example, suppose a fixation process produces events that are not a true representation of the fixed cells being assayed in your samples, and are highly fluorescent. It is important to identify and exclude these events in your unstained control files before running Resolve, and to exclude spectra associated with these events from your single stain controls. This allows Resolve to estimate fluorescent and autofluorescent properties of relevant cellular events. 

Thus, when possible, it is optimal to include unstained and  controls that are as similar as possible to the experimental files you plan to assay. Such steps can include:

  • Using controls processed through the exact same collection method
  • Collecting controls following the same experimental steps as you intend to follow for samples stained by the complete panel
  • Including unstained controls for each biological matrix you intend to assay

Later in this document, you will learn about the spectral filtering tool available in Resolve. This feature enables using complex material types in the Resolve workflow. Because of this tooling, we recommend using cellular controls when possible, even for highly rare markers, since unmixing can proceed so long as you are able to identify events (even just a handful) that have taken up the staining fluor through the spectral filtering workflow.

Appendix C: Keyboard Shortcuts

Gating Interface:

  • Shift + scroll: Resize gates
  • Shift + click/drag: Rotate gates
  • Double-click: Add vertex
  • Right-click: Context menu

Appendix D: Recommended Workflows

  • Small Panels (≤8 colors): Manual metadata entry acceptable
  • Large Panels (>8 colors): CSV uploads strongly recommended

Appendix E: Exporting Data and Importing into other Platforms

OMIQ

  • After setting up a Workflow, open up a ‘Scales’ task.
  • In the ‘Bulk Edit’ window, select all fluorescent parameters and choose Arcsinh as the Scaling Type
  • Set the Display Bounds to a Minimum of 0, and a Maximum of 3,000,000
  • Click Apply
  • In the main ‘Scales’ task window, type in the optimized cofactor values from the exported .csv file provided by Ozette Resolve.
  • Save and resume analysis in the Workflow.

FlowJo Version 10 (Note: ArcSinh not supported in Version 11 at this time)

  • Add Ozette Resolve unmixed files to a new workspace
  • Double click on a file to open a Graph window
  • Change either axis to a fluorescent parameter and click the ‘T’ next to the parameter and select ‘Customize Axis’
  • Select all fluorescent parameters in the panel on the right hand side.
  • Adjust the fluorescent scale to view the full range by clicking the ‘+’ sign on the right hand side of the histogram.
    • This should be just past 10^6 for Cytek Aurora data
  • Select ‘ArcSinh’ from the scale drop down menu.
  • Change the ‘Width Basis’ to -1000 and hit apply. This will provide a good starting point for assigning cofactors for each fluor.
  • Reopen the ‘Customize Axis’ window.
  • Select the parameter displayed on the histogram and type in the optimized cofactor from the exported .csv file provided by Ozette Resolve. Click Apply.
  • Change the axis in the graph window to the next fluorescent parameter and repeat the process for all fluorescent parameters.
  • Save workspace.

FCS Express

  • Go to File > Options.
  • In the Options window, go to: User Options > Data Analysis > Scaling
  • Locate the settings for default scaling (for X and Y axes).
  • Select arcsinh as the default scaling type.
  • Type in the optimized cofactors for each fluorescent parameter from the exported .csv file provided by Ozette Resolve.
  • Click OK to save