Evaluation of Marker Resolution

The video introduces the marker resolution evaluation protocol for high-dimensional immunophenotyping. Open all single-stained cell controls and the full-stain sample in data analysis software (FlowJo v10 or equivalent).

Gate the sample by time to remove unstable flow events. Remove doublets by plotting forward scatter area versus height, then repeat using side scatter to isolate singlet populations.

Remove dead cells using a viability gate applied to the single-stained samples, adjusting gates as needed for the fully-stained sample. Isolate cells of interest by plotting forward scatter versus side scatter and gating on the lymphocyte population.

Open the layout editor and create histograms for each single-stain control marker using the cleaned lymphocyte population. Ensure the x-axis parameter matches each sample's fluorophore label (e.g., BV650).

Convert histograms to dot plots for better visibility of rare positive populations. Drag the full-stain sample's lymphocyte population onto each single-stain dot plot to create overlays for direct comparison.

Inspect each overlay to assess resolution loss between positive and negative populations. Check whether the negative population broadens in the full-stain sample compared to the single-stain, and confirm this broadening does not impair gating ability.

Examine whether positive population intensity is reduced in the full-stain compared to single-stain samples. Determine if staining reduction is problematic or if titration adjustments or sequential staining strategies are needed.

Conclude the resolution evaluation and determine if the panel is acceptable without further changes. Note that autofluorescence extraction techniques can be applied to improve resolution, which will be covered in the next video.