TY - JOUR
T1 - Secretion of insulin-like growth factor binding protein-1 from individual hepatocytes
AU - Eghbali-Fatourechi, G.
AU - Conover, C. A.
AU - Sieck, G. C.
AU - Gores, G. J.
AU - Fitzpatrick, L. A.
PY - 1994
Y1 - 1994
N2 - 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.
AB - 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.
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M3 - Article
C2 - 7530114
AN - SCOPUS:0027971143
SN - 0034-5164
VL - 85
SP - 243
EP - 259
JO - Research Communications in Molecular Pathology and Pharmacology
JF - Research Communications in Molecular Pathology and Pharmacology
IS - 3
ER -