Precision Vaccines Program, Division of Infectious Diseases, Boston Children's Hospital, Boston, MA, USA.
Precision Vaccines Program, Division of Infectious Diseases, Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
STAR Protoc. 2023 Sep 15;4(3):102405. doi: 10.1016/j.xpro.2023.102405. Epub 2023 Jul 13.
High-throughput screening is a powerful platform that can rapidly provide valuable 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 cytometry. Cryopreserved PBMCs are incubated for 72 h with various small molecule libraries 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 immunomodulators and potential vaccine adjuvant candidates. For complete details on the use and execution of this protocol, please refer to Chew et al..
高通量筛选是一种强大的平台，可以快速为数千种化合物提供有价值的细胞毒性、免疫学和表型信息。在自体血浆中培养的人外周血单核细胞 (PBMC) 可以模拟人类的免疫反应。在这里，我们描述了一种方案，即用该方案刺激 PBMC 培养 72 小时，并通过 AlphaLISA 分析检测细胞因子的分泌，通过流式细胞术检测细胞表面激活标志物的表达。将冷冻保存的 PBMC 与各种小分子文库孵育 72 小时，收集上清液，通过 AlphaLISA 分析快速测量关键细胞因子（肿瘤坏死因子-α、干扰素-γ、白细胞介素 10）的分泌水平。几乎同时，细胞可以用针对固有免疫激活标志物（CD80、CD86、HLA-DR、OX40）的抗体固定和染色，然后通过流式细胞术进行分析。这种多重读出工作流程可以直接帮助鉴定和发现新的免疫调节剂和潜在的疫苗佐剂候选物。有关该方案的使用和执行的完整详细信息，请参阅 Chew 等人的研究。
