Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies to Enable Combination Multiplexing.

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 intensities 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 and fluorescence correlation spectroscopy showed that the ∼3-fold brightness difference is due to static quenching most likely by the oligo or solution in the ext. configuration. Spectral flow cytometry experiments using peripheral blood mononuclear cells show int. MuSIC probe-labeled antibodies (i) retained increased signal intensity while having no significant difference in the estimated % of CD8+ lymphocytes and (ii) labeled with Atto488, Atto647, and Atto488/647 combinations can be demultiplexed in triple-stained samples. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications where spectral detection is available.

Method for Improved Fluorescence Corrections for Molar Mass Characterization by Multiangle Light Scattering.

Multiangle light scattering (MALS) was used to determine the absolute molar mass of fluorescent macromolecules. It is standard protocol to install bandwidth filters before MALS detectors to suppress detection of fluorescent emissions. Fluorescence can introduce tremendous error in light scattering measurements and is a formidable challenge in accurately characterizing fluorescent macromolecules and particles. However, we show that for some systems, bandwidth filters alone are insufficient for blocking fluorescence in molar mass determinations. For these systems, we have devised a correction procedure to calculate the amount of fluorescence interference in the filtered signal. By determining the intensity of fluorescent emission not blocked by the bandwidth filters, we can correct the filtered signal accordingly and accurately determine the true molar mass. The transmission rates are calculated before MALS experimentation using emission data from standard fluorimetry techniques, allowing for the characterization of unknown samples. To validate the correction procedure, we synthesized fluorescent dye-conjugated proteins using an IR800CW (LI-COR) fluorophore and Bovine Serum Albumin protein. We successfully eliminated fluorescence interference in MALS measurements using this approach. This correction procedure has potential application toward more accurate molar mass characterizations of macromolecules with intrinsic fluorescence, such as lignins, fluorescent proteins, fluorescence-tagged proteins, and optically active nanoparticles.

Protocol for Creating Antibodies with Complex Fluorescence Spectra.

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.

Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies.

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.

Theory for High-Throughput Genetic Interaction Screening.

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most ∼100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose ∼105 spectrally unique, genetically encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.