Elevated Autofluorescence in Myeloid and Granular Cells (Flow Cytometry (Sample Considerations))

Question

Accepted Answer

High background fluorescence detected in shorter wavelength channels (BV421, FITC, PE), particularly in larger granular cells such as monocytes, neutrophils, eosinophils, macrophages, and dendritic cells, compromising signal-to-noise ratio for true positive events. Common causes: 1) Biological structures (mitochondria, lysosomes) emit natural fluorescence captured in shorter wavelength channels (BV421, FITC, PE) 2) Intracellular fluorescent compounds (NADPH) accumulate in metabolically active and granular myeloid cells 3) Cell size and granularity directly correlate with autofluorescence intensity; larger myeloid cells exhibit increased background 4) Unstained controls reveal differential autofluorescence: eosinophils > neutrophils > monocytes > lymphocytes in FITC channel Solutions: 1) Assign dim antigens to longer wavelength fluorophores (APC, APC-Cy7) where autofluorescence is minimal 2) Reserve shorter wavelength channels (BV421, FITC, PE) for highly expressed markers to overcome autofluorescence background 3) Include unstained samples from each cell type (lymphocytes, monocytes, neutrophils, eosinophils) to measure baseline autofluorescence per population 4) Apply autofluorescence extraction algorithms or subtract autofluorescence controls during analysis to correct for background