Autofluorescence extraction on the CytoFLEX mosaic Spectral Detection Module 9420

Abstract Description Cells exhibit an inherent fluorescence called autofluorescence (AF) caused by cellular components and metabolites that fluoresce upon excitation by specific wavelengths of light. Common endogenous fluorophores include cyclic compounds such as reduced pyridine nucleotides (NADH), oxidized flavin coenzymes (FMN and FAD), vitamins, and proteins containing aromatic amino acids like tryptophan, phenylalanine and tyrosine. The analysis of flow cytometry data can be complicated by the presence of cells intrinsic AF: interfering with the precise detection of the desired fluorescence signals, particularly for low-abundance markers or dim populations. In addition, AF can lead to unmixing errors. To test AF extraction on the CytoFLEX mosaic spectral detection module, we stained a 100 ul whole blood sample with a combination of fluorochromes that are either within or outside of the expected AF range of white blood cells. Samples were acquired using the CytoFLEX mosaic spectral detection module. During unmixing, 4 AF populations could be identified corresponding with different spectra and fluorescent signatures. After removing highly similar AF spectra, 2 unique signatures remained. Including these 2 unique AF spectra in the unmixing increased the resolution for the fluorochromes within the expected AF range. In conclusion, this work demonstrates the effectiveness of using multiple AF signatures for AF extraction on the CytoFLEX mosaic spectral detection module in complex samples. Funding Sources N/A Topic Categories Technological Innovations in Immunology (TECH)