Quantifying Immune Effector Molecules from Mouse Leukocytes with Flow-Based Multiplex Assay Panels

Abstract

Abstract Exposure to foreign material triggers host immune responses, which are primarily mediated by leukocytes including macrophages, neutrophils, dendritic cells, NK cells, T cells and B cells. These cells produce an array of soluble factors which coordinate host defense, tissue repair, and immunoregulatory functions. In addition, these cells and the factors they produce are also critically involved in various diseases including infection, autoimmune disorders, asthma, diabetes, and cancer. Therefore, quantification of these molecules in a cell type-specific context will help achieve in-depth understanding of the immune responses and disease processes. We have developed multiplex assay panels targeting specific mouse immune cells (CD4+ T helper cells, Macrophages/Microglia and B cells), using fluorescence–encoded beads that are suitable for use on common lab flow cytometers. Each panel allows for simultaneously quantification of 13 related analytes as shown in the table below. Cell TypePanel TargetsT helper cellsIL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, TNF-αMacrophages/MicrogliaIL-1β, 6, 10, 12p40, 12p70, 18, 23, TNF-α, CCL22/MDC, CCL17/TARC, CXCL1/KC, G-CSF, TGF-β1B cellsIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, BAFF, BCMA, sCD40 ligand, TGF-β1 Each antibody pair was carefully optimized for assay specificity, sensitivity, accuracy and reproducibility. In addition, these panels have been validated by detecting expected expression level changes of analytes in biological samples cultured under various stimulation conditions. These high quality, low cost and easy to use panels will be useful tools to the study of specific cells involved in innate and adaptive immune responses and the relevant diseases.