Optimization of the Blocking and Signal Preservation Protocol in High-Parameter Flow Cytometry

High quality input data is the key to successful interpretation of any scientific assay. In flow cytometry, fluorescently-conjugated antibodies allow us to simultaneously measure an incredible range of protein-based targets on single cells with a high degree of specificity. Limiting the quality of data generated, however, is the non-specific interaction that can occur between antibodies and off-target binders. Judicious use of blocking reagents can improve the specificity of the staining by reducing this non-specific binding to cells, improving the sensitivity of the assay to detect the authentic signal above assay noise. Additional beneficial effects include preventing interactions between dyes and even limiting the degradation of dyes, improving data quality. In this article, we provide a workflow for minimizing these unwanted effects, increasing specificity and sensitivity in highly multiplex flow cytometry. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Surface staining Basic Protocol 2: Intracellular staining Basic Protocol 3: Intracellular cytokine staining.