Cyanine TruStain™, an effective blocking buffer to eliminate non-specific cyanine-like dye-mediated monocyte binding

Abstract

Abstract Cyanine dyes and their analogs are commonly used as acceptors in commercially available antibody-tandem fluorophore conjugates. However, it has been confirmed that the dyes have a tendency to bind monocytes or macrophages nonspecifically, which limits the ability to do multi-color flow cytometric analysis for lower density antigens. We are introducing Cyanine TruStain™ Buffer which eliminates this nonspecific binding. This reagent blocks nonspecific binding of monocytes and macrophages to Cyanine dyes, (PE/Cy7, PE/Cy5, PercpCy5.5, APC/Cy7, APC/Fire™ 750, PE/Dazzle™ 594). It does not affect binding of, e.g., CD64, CD14 and other tested antibodies. Multiple Cyanine dyes can be blocked at once and stability data shows no decrease in signal intensity compared to the conjugate alone. In addition the reagent has no impact on cell viability. Mock sorting experiments were performed with PBMC to determine if the Cyanine TruStain™ Buffer stimulates cells to produce cytokines and chemokines. Our data indicates minimal to no stimulation above conjugated antibody alone for IL-6, IL-8, IL-10 and MCP-1 production. We also tested Cyanine TruStain™ Buffer for its ability to negatively affect PBMC proliferation in response to anti-CD3/anti-CD28 stimulation. We observed minimal to no effect on proliferation by measuring BrdU incorporation. In summary, we are introducing a blocking reagent to eliminate nonspecific binding to Cyanine dyes which shows minimal perturbation of cell functions.