Why did my Flow cytometry fail?
Target fluorophore signal too low to resolve positive from negative populations. Spectral spillover not correctly compensated — populations skewed or negative MFI artifacts. Elevated non-specific fluorescence obscuring true positives.
Playbooks
- Flow cytometry: weak signalTarget fluorophore signal too low to resolve positive from negative populations.
- Flow cytometry: compensation issueSpectral spillover not correctly compensated — populations skewed or negative MFI artifacts.
- Flow cytometry: high backgroundElevated non-specific fluorescence obscuring true positives.
Calculators
FAQ — Flow cytometry
- Weak signal
- Target fluorophore signal too low to resolve positive from negative populations. Titrate the antibody on positive-control cells (e.g. 1:50 → 1:200) — Separates under-titration from instrument settings in one plate. Raise PMT voltage using single-stained controls until positives clear the negative — Under-voltage is a common silent change after a shared cytometer session. Extend staining time to the validated window (e.g. 30–45 min protected from light) — Incomplete epitope engagement mimics a dead conjugate.
- Compensation issue
- Spectral spillover not correctly compensated — populations skewed or negative MFI artifacts. Rebuild compensation with single-stained controls matched to experimental brightness — Most spillover artifacts are from mismatched compensation controls. Verify optical configuration matches the panel; re-run auto-compensation after service — Laser/filter changes invalidate old matrices. Re-titrate a new conjugate lot and rebuild the matrix before the next multi-colour run — Brightness changes shift required spillover coefficients.
- High background
- Elevated non-specific fluorescence obscuring true positives. Add Fc block (or serum block) before staining; include FMO and isotype controls — Separates non-specific binding from true antigen signal. Include a viability dye and gate out dead cells / debris — Dead cells bind antibodies non-specifically and inflate background. Titrate antibody down 2-fold on FMO-controlled samples — Over-titration is a common material cause of high background.