Fluorescent Antibody Multiplexing with Oligo-Based Combinatorial Labeling

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, 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, which show a degree of labeling of ~9.66 linker molecules / antibody. 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 incubated MuSIC probe-labeled antibodies with protein A beads to create single and double positive beads that are analogous to single cells. 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 is accurately measured. 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. ### Competing Interest Statement The authors have declared no competing interest.