Department of Immunology, School of Medicine, UConn Health.
Center for Molecular Discovery, University of New Mexico Health Sciences Center.
J Vis Exp. 2024 Feb 2(204). doi: 10.3791/64401.
This protocol aims to establish a method for identifying small molecular antagonists of β2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. β2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling β2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of β2 integrins, is utilized to quantify β2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil β2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on β2 integrin inhibition are assessed within 3 h. Molecules that directly target β2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.
本方案旨在建立一种利用构象变化报告型抗体和高通量流式细胞术鉴定β2 整合素激活小分子拮抗剂的方法。该方法也可作为其他基于抗体的高通量筛选方法的指南。β2 整合素是白细胞特异性黏附分子,在免疫反应中至关重要。中性粒细胞依赖整合素激活离开血液,不仅是为了抗感染,还参与多种炎症性疾病。控制β2 整合素激活为治疗与中性粒细胞相关的炎症性疾病提供了一种可行的方法。在本方案中,使用一种单克隆抗体 mAb24,它特异性结合β2 整合素的高亲和力头部,来定量分离的原代人中性粒细胞上β2 整合素的激活。N-甲酰基-甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)被用作刺激物来激活中性粒细胞的β2 整合素。本研究使用了一种能够自动运行 384 孔板样本的高通量流式细胞仪。在 3 小时内评估了 320 种化学物质对β2 整合素抑制的影响。通过这种方法可以鉴定直接靶向β2 整合素的分子或靶向 G 蛋白偶联受体起始的整合素内-外激活信号通路中的分子。