Autofluorescence Interferes with Detection Channels (Flow Cytometry (Sample Considerations))

Question

Accepted Answer

High background fluorescence observed in BV421, FITC, and PE channels, particularly with myeloid cells (monocytes, macrophages, neutrophils, eosinophils). Positive signal difficult to distinguish from cellular autofluorescence. Common causes: 1) Natural fluorescence from mitochondria, lysosomes, and NADPH emits in shorter wavelength channels (BV421, FITC, PE) 2) Larger and more granular myeloid cells contain elevated levels of intracellular fluorescent compounds 3) Cell size, metabolic activity, and granularity increase autofluorescence intensity 4) Neutrophils and eosinophils show particularly high autofluorescence in FITC channel Solutions: 1) Avoid assigning dim antigens to BV421, FITC, and PE channels when analyzing myeloid-rich samples 2) Place bright markers or highly expressed antigens in autofluorescence-prone channels 3) Include unstained controls for each cell subset to quantify baseline autofluorescence 4) Consider using longer wavelength fluorophores (APC, AF647, AF700) for dim antigens on granular cells 5) Use autofluorescence extraction algorithms during data analysis if available