Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, 07740 Jena, Germany; Center for Molecular Biomedicine, Jena University Hospital, 07745 Jena, Germany; Center for Sepsis Control and Care, Jena University Hospital, 07740 Jena, Germany.
Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, 07740 Jena, Germany; Center for Molecular Biomedicine, Jena University Hospital, 07745 Jena, Germany.
J Immunol Methods. 2021 Mar;490:112953. doi: 10.1016/j.jim.2020.112953. Epub 2020 Dec 30.
The sphingosine 1-phosphate receptor type 1 (S1PR1) has several important functions, including stabilizing endothelial barrier and maintaining lymphocyte circulation. These functions are critically dependent on the regulation of S1PR1 cell surface expression. Currently available antibodies against human S1PR1 are not able to pick up cell surface expression on living cells by flow cytometry due to intracellular epitopes or unspecific binding. Here we describe the generation of a mouse monoclonal antibody specific for the N-terminal region of human S1PR1. It has an immunoglobulin M (IgM) kappa isotype and detects cell surface expression of recombinant human S1PR1 on overexpressing cells. Due to unspecific intracellular cell staining, it cannot be used for staining of dead cells and tissue slides or in microscopic analyses. It is also not suitable for Western blot analysis and immunoprecipitation. However, the antibody can stain for endogenous S1PR1 on human endothelial cell lines and primary human umbilical vein endothelial cells (HUVEC). Incubation of these cells with various S1PR1 agonists revealed potent S1PR1 internalization, which was not the case with the specific antagonist W146. Surprisingly, human T and B cells isolated from blood and palatine tonsils did not show specific staining, demonstrating significantly lower endogenous S1PR1 surface expression on lymphocytes than on endothelial cells.
S1P 受体 1（S1PR1）具有多种重要功能，包括稳定内皮屏障和维持淋巴细胞循环。这些功能取决于 S1PR1 细胞表面表达的调节。目前可用于流式细胞术检测活细胞表面 S1PR1 的抗体由于细胞内表位或非特异性结合而无法识别。本研究描述了一种针对人 S1PR1 N 端区域的小鼠单克隆抗体的产生。它是一种免疫球蛋白 M（IgM）κ 同种型，可检测过表达细胞中重组人 S1PR1 的细胞表面表达。由于细胞内非特异性染色，该抗体不能用于死细胞和组织切片的染色或显微镜分析。它也不适合用于 Western blot 分析和免疫沉淀。然而，该抗体可用于染色人内皮细胞系和原代人脐静脉内皮细胞（HUVEC）中的内源性 S1PR1。用各种 S1PR1 激动剂孵育这些细胞可引发 S1PR1 内吞，而特异性拮抗剂 W146 则没有这种作用。令人惊讶的是，从血液和腭扁桃体分离的人 T 和 B 细胞未显示出特异性染色，表明淋巴细胞上的内源性 S1PR1 表面表达明显低于内皮细胞。
