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.

1 型鞘氨醇 1-磷酸受体 (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。这些功能严重依赖于 S1PR1 细胞表面表达的调节。由于细胞内表位或非特异性结合，目前可用的抗人 S1PR1 抗体无法通过流式细胞术检测活细胞上的细胞表面表达。在这里，我们描述了对人类 S1PR1 的 N 末端区域具有特异性的小鼠单克隆抗体的生成。它具有免疫球蛋白 M (IgM) kappa 同种型，可检测重组人 S1PR1 在过表达细胞上的细胞表面表达。由于非特异性细胞内细胞染色，它不能用于死细胞和组织切片的染色或显微镜分析。它也不适合蛋白质印迹分析和免疫沉淀。然而，该抗体可以对人内皮细胞系和原代人脐静脉内皮细胞 (HUVEC) 上的内源性 S1PR1 进行染色。这些细胞与各种 S1PR1 激动剂一起孵育显示出有效的 S1PR1 内化，而特异性拮抗剂 W146 则不然。令人惊讶的是，从血液和腭扁桃体中分离的人 T 和 B 细胞没有显示出特异性染色，表明淋巴细胞上的内源性 S1PR1 表面表达明显低于内皮细胞。这些细胞与各种 S1PR1 激动剂一起孵育显示出有效的 S1PR1 内化，而特异性拮抗剂 W146 则不然。令人惊讶的是，从血液和腭扁桃体中分离的人 T 和 B 细胞没有显示出特异性染色，表明淋巴细胞上的内源性 S1PR1 表面表达明显低于内皮细胞。这些细胞与各种 S1PR1 激动剂一起孵育显示出有效的 S1PR1 内化，而特异性拮抗剂 W146 则不然。令人惊讶的是，从血液和腭扁桃体中分离的人 T 和 B 细胞没有显示出特异性染色，表明淋巴细胞上的内源性 S1PR1 表面表达明显低于内皮细胞。