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• W3161154649 abstract "Fluorescent antibodies are a workhorse of biomedical science, but fluorescence multiplexing has been notoriously difficult due to spectral overlap between fluorophores. We recently established proof-of-principal for fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC), which uses combinations of existing fluorophores to create unique spectral signatures for increased multiplexing. However, a method for labeling antibodies with MuSIC probes has not yet been developed. Here, we present a method for labeling antibodies with MuSIC probes. We conjugate a DBCO-Peg5-NHS ester linker to antibodies and a single-stranded DNA docking strand to the linker and, finally, hybridize two MuSIC-compatible, fluorescently labeled oligos to the docking strand. We validate the labeling protocol with spin-column purification and absorbance measurements. We demonstrate the approach using (i) Cy3, (ii) Tex615, and (iii) a Cy3-Tex615 combination as three different MuSIC probes attached to three separate batches of antibodies. We created single-, double-, and triple-positive beads that are analogous to single cells by incubating MuSIC probe-labeled antibodies with protein A beads. Spectral flow cytometry experiments demonstrate that each MuSIC probe can be uniquely distinguished, and the fraction of beads in a mixture with different staining patterns are accurately inferred. The approach is general and might be more broadly applied to cell-type profiling or tissue heterogeneity studies in clinical, biomedical, and drug discovery research." @default.
• W3161154649 created "2021-05-24" @default.
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• W3161154649 date "2021-05-19" @default.
• W3161154649 modified "2023-09-27" @default.
• W3161154649 title "Protocol for Creating Antibodies with Complex Fluorescence Spectra" @default.
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• W3161154649 doi "https://doi.org/10.1021/acs.bioconjchem.1c00220" @default.
• W3161154649 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/34009954" @default.
• W3161154649 hasPublicationYear "2021" @default.
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