Autofluorescence Interfering with Viability Dye Detection (Flow Cytometry (Autofluorescence))

Question

Accepted Answer

Dead cell discrimination becomes unreliable as autofluorescence overlaps with viability dye emission spectra. False positive or false negative viability calls occur, particularly with green or orange viability dyes. Common causes: 1) Spectral overlap between cellular autofluorescence and viability dye emission 2) FITC or PE-based viability dyes highly susceptible to autofluorescence interference 3) Autofluorescent cell types (monocytes, activated cells) masking viability signal 4) UV and blue laser excitation inducing strong autofluorescence in viability channels Solutions: 1) Select viability dyes in far-red channels (e.g., 633 nm or 780 nm) to avoid autofluorescence 2) Validate viability gating with FMO controls excluding viability dye 3) Include heat-killed cell controls to establish dead cell gating parameters 4) Avoid FITC-based viability dyes (e.g., 7-AAD alternatives) in autofluorescent samples 5) Use spectral unmixing to separate viability dye signal from autofluorescence