Unmixing Evaluation of Fully Stained Sample

Place time on the x-axis and gate out any events collected during unstable flow conditions such as bubbles or clogs. This sample shows clean collection with minimal exclusion needed.

Gate out doublets by plotting forward scatter area versus height, selecting the diagonal singlet population. Then repeat the process using side scatter area versus height to further isolate single cells.

Review n-by-n plots created in the previous protocol to identify super-bright aggregate events that may be incorrectly unmixed. In this clean sample, no aggregates were detected during processing.

Plot viability dye on the x-axis versus side scatter and gate on events negative for the viability dye while excluding low forward scatter debris. This produces a clean population of viable single cells.

Gate on the lymphocyte population within the viable single cell population, as this panel is focused on T cell analysis. All subsequent analysis should be performed on this gated population.

Create a plot for each marker in the panel with the marker on the x-axis and side scatter on the y-axis. Scan through all plots to confirm positive signal is present for every antibody, even if the signal is rare or dim.

Review the worksheet template with two-dimensional plots for each marker in the fully stained sample. Verify that positive populations show centered distributions without skewing and that no unmixing errors are apparent, proceeding to the next protocol if results look correct.