Evaluation of Optimal Spectral Reference Controls

Examine the forward and side scatter plots of unstained cells and beads to confirm all populations are on scale and cleanly separated. Check that forward scatter area versus height shows a 45-degree diagonal pattern, confirming optimal FSC scaling and enabling singlet detection.

Review the instrument settings for fluorescent detector gains used during acquisition and cross-reference them against the quality control report from the day of sample collection. Verify that gains are in the same ballpark across all lasers and all experimental tubes.

Generate a spectral plot of the unstained sample and examine it for any distinct fluorescence signatures beyond expected autofluorescence. Confirm that the unstained control shows only background autofluorescence with no evidence of dye contamination.

Open the unmixing wizard with all bead-stained controls and create precise gates on single bead populations. For each fluorophore, verify that the peak channel matches expected specifications, confirm the full spectral signature matches reference data, and ensure tight event populations with no contaminating peaks.

Check each fluorescent control's negative population by dragging the positive gate to the negative region and confirming no signal carryover or accidental staining. Ensure all negative controls are truly unstained with no cross-contamination between tubes.

Navigate to the QC controls tab and review the similarity matrix to confirm each dye's spectral signature is sufficiently unique. Verify that all similarity index values are 0.98 or below, confirming the instrument can resolve all dyes in the panel.

Execute live unmixing using bead controls and then examine the unmixed single-stained cell samples to assess whether bead controls are sufficient. If spectral mismatches are detected in cells, prepare to rerun unmixing using cell-based controls instead.