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• W4383550906 abstract "While full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensity than conventionally-labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5' or 3' end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a ∼6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence spectroscopy and fluorescence correlation spectroscopy showed that ∼3-fold brightness difference is due to static quenching. Spectral flow cytometry experiments using peripheral blood mononuclear cells stained with anti-CD8 antibodies showed that int. MuSIC probe-labeled antibodies have signal intensity equal to or greater than conventionally-labeled antibodies with similar estimated proportion of CD8+ lymphocytes. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications." @default.
• W4383550906 created "2023-07-08" @default.
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• W4383550906 date "2023-07-06" @default.
• W4383550906 modified "2023-09-27" @default.
• W4383550906 title "Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies" @default.
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• W4383550906 doi "https://doi.org/10.1101/2023.07.06.547965" @default.
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