Figure 1

Combinatorial immunostainings used for the identification of bovine immune cell subsets. The staining procedure used a seven-step, eleven-color assay. Three panels were designed for in depth characterization of antigen presenting cells (i.e., monocytes, macrophages and DCs) (A), T cells (CD4⁺, CD8⁺ and γδ T cells) (B) and B and NK cells (C). The Similarity Index is a measure of dye pair uniqueness on a scale from 0 (the 2 dyes are very divergent from each other) to 1 (the 2 dyes are very similar to each other), indicating that none of our dyes were incompatible with each other (acceptable threshold ≤ 0.98). The Complexity Index is an overall measure of uniqueness of all dyes in a full spectrum cytometry panel. Our three eleven-color combinations have a complexity Index < 3, proving they were well-designed panels according to the manufacturer. Emission channel illustrations are taken from Cytek Full Spectrum Viewer website. ^aDue to product discontinuation, the antibody combination panel was occasionally slightly modified: IgG1-AF405 and IgG2a-PE-C7 rather than IgG1-PE-C7 and IgG2a-SB436, respectively. ^bLabeled with Zenon™ Mouse IgG1 Labeling Kit, AF647. ^cLabeled with AF532 Antibody Labeling Kit. ^dLabeled with Zenon™ Mouse IgG1 Labeling Kit, AF700.