Eghbali-Fatourechi G, Conover C A, Sieck G C, Gores G J, Fitzpatrick L A
Department of Anesthesiology, Mayo Clinic, Rochester, MN 55905.
Res Commun Mol Pathol Pharmacol. 1994 Sep;85(3):243-59.
The reverse hemolytic plaque assay (RHPA) uses complement-mediated red blood cell lysis to detect peptide secretion by individual cells. Initially, the RHPA was used to study the function of neurons and B lymphocytes. More recently, the RHPA has been adapted to measure hormone release from individual pituitary, parathyroid, luteal, and pancreatic islet cells. We have applied this technique to detect insulin-like growth factor binding protein-1 (IGFBP-1) secretion by a human hepatoma cell line (HepG2). We proposed that the technique of RHPA could be used to study peptide release from single hepatocytes in various defined conditions. Our goal was the study of the kinetics of IGFBP-1 secretion from hepatoma cells and rat hepatocytes and to determine the heterogeneity of the cell population regarding the secretion of IGFBP-1. To evaluate the optimal conditions of IGFBP-1 secretion by hepatoma cells and rat hepatocytes and to evaluate the influence of cell dispersion on hepatocyte's behavior, we evaluated three techniques of cell dispersion: trypsin digestion, collagenase digestion, and mechanical dispersion. We tested cell viability, determined the percentage of secreting cells versus non-secreting cells, and measured mean plaque area which is a function of the amount of IGFBP-1 secreted by an individual cell. We determined the optimal IGFBP-1 antibody dilution for the detection of secreted IGFBP-1 by hepatocytes, evaluated the initiation of IGFBP-1 secretion from cultured cells, and quantified time-dependent IGFBP-1 secretion. In addition to demonstrating the feasibility of measuring IGFBP-1 from a cultured cell line, we measured IGFBP-1 release from freshly dispersed rat hepatocytes.
反向溶血空斑试验（RHPA）利用补体介导的红细胞裂解来检测单个细胞的肽分泌。最初，RHPA用于研究神经元和B淋巴细胞的功能。最近，RHPA已被用于测量单个垂体、甲状旁腺、黄体和胰岛细胞的激素释放。我们已应用该技术检测人肝癌细胞系（HepG2）分泌的胰岛素样生长因子结合蛋白-1（IGFBP-1）。我们提出，RHPA技术可用于研究在各种特定条件下单个肝细胞的肽释放。我们的目标是研究肝癌细胞和大鼠肝细胞中IGFBP-1分泌的动力学，并确定细胞群体在IGFBP-1分泌方面的异质性。为了评估肝癌细胞和大鼠肝细胞分泌IGFBP-1的最佳条件，并评估细胞分散对肝细胞行为的影响，我们评估了三种细胞分散技术：胰蛋白酶消化、胶原酶消化和机械分散。我们测试了细胞活力，确定了分泌细胞与非分泌细胞的百分比，并测量了平均空斑面积，其是单个细胞分泌的IGFBP-1量的函数。我们确定了用于检测肝细胞分泌的IGFBP-1的最佳IGFBP-1抗体稀释度，评估了培养细胞中IGFBP-1分泌的起始，并对时间依赖性IGFBP-1分泌进行了定量。除了证明从培养细胞系中测量IGFBP-1的可行性外，我们还测量了新鲜分散的大鼠肝细胞中IGFBP-1的释放。
