FITC Green Channel Highly Affected by Autofluorescence (Flow Cytometry (Autofluorescence))

Question

Accepted Answer

FITC-conjugated antibodies show poor signal-to-noise ratio with elevated background in green channel. Blue laser (488 nm) excitation induces strong autofluorescence that directly overlaps with FITC emission spectrum. Common causes: 1) FITC emission directly overlaps with NADH and flavin autofluorescence emissions 2) Blue laser (488 nm) inducing strongest cellular autofluorescence 3) UV laser (355 nm) also contributing to green channel autofluorescence 4) FITC classified as highly susceptible dye to autofluorescence interference Solutions: 1) Avoid FITC for critical markers; select alternative fluorophores (PE, APC, BV421) 2) Use dyes with emissions outside autofluorescence range for important antigens 3) Implement spectral unmixing to separate FITC signal from NADH/flavin autofluorescence 4) Reserve FITC for high-expression markers only where signal overwhelms background 5) Switch to red/far-red lasers and dyes (633 nm and beyond) for least autofluorescence interference