Flow cytometry: compensation issue

1. Ranked causes

   1. Compensation controls missing, wrong fluorophore, or too dim/bright vs experimentSingle-stained controls do not match experimental brightness or fluorophore.
   2. Antibody lot brightness changed (new conjugate needs re-compensation)New vial is much brighter/dimmer than the matrix was built on.
   3. Gating on the wrong population when building the matrixRebuilding compensation on correctly gated single stains fixes skew.
   4. Auto-compensation matrix outdated after filter / laser changeIssue started after cytometer service or optical change.

2. Recovery steps

   1. Rebuild compensation with single-stained controls matched to experimental brightnessMost spillover artifacts are from mismatched compensation controls.
   2. Verify optical configuration matches the panel; re-run auto-compensation after serviceLaser/filter changes invalidate old matrices.
   3. Re-titrate a new conjugate lot and rebuild the matrix before the next multi-colour runBrightness changes shift required spillover coefficients.

3. Controls first

   • Were single-stained compensation controls run with the same lots and brightness as the panel?If no: Rebuild compensation before interpreting multi-colour results.
   • Did the positive single-stain control show the expected fluorophore peak?If no: Control stain or lot failure — fix controls before touching the matrix.

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