A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

High-throughput screening is a powerful platform that can rapidly provide valu-able cytotoxic, immunological, and phenotypical information for thousands of compounds. Human peripheral blood mononuclear cells (PBMCs) cultured in autologous plasma can model the human immune response. Here, we describe a protocol to stimulate PBMCs for 72 h and measure cytokine secretion via AlphaLISA assays and cell surface activation marker expression via flow cytome-try. Cryopreserved PBMCs are incubated for 72 h with various small molecule li-braries and the supernatants are harvested to rapidly measure secretion levels of key cytokines (tumor necrosis factor alpha, interferon gamma, interleukin 10) via the AlphaLISA assay. Almost simultaneously, the cells can be fixated and stained using antibodies against innate immune activation markers (CD80, CD86, HLA-DR, OX40) for analysis via flow cytometry. This multiplexed readout workflow can directly aid in the phenotypic identification and discovery of novel immuno-modulators and potential vaccine adjuvant candidates. For complete details on the use and execution of this protocol, please refer to Chew et al.1