Assay of ligand receptors on membranes of epithelial cells cultured in three dimensional matrices

Abstract

SUMMARY: Three-dimensional matrix culture procedures are gaining favor for analysis of epithelial cell function. However, it is difficult to determine the kinetics of ligand binding to cells cultured within a matrix. We have developed a procedure that allows analysis of ligand binding to mouse mammary epithelial cells cultured in collagen gets using membrane vesicles. Cells are first recovered from collagen gels by digestion with collagenase and subsequently washed with sucrose solution. The cells in suspension are then disrupted and membranes are isolated by two sequential centrifugations. These cell membranes are used for determination of receptor binding kinetics and the nuclear pellet is used to estimate the DNA content. This method circumvents the disadvantages of the inaccessibility of receptor sites on cells cultured inside the matrix. The procedures are adaptable to various cell types and matrix materials.

Membrane fragments from cultured endothelial cells for use in screening anti-FGF receptor antibodies

“…The preparation of celt surface membranes in a form as pure as possible from other cell organelles is a new approach to the isolation and characterization of specific antigens, which could be used to produce or screen antibodies or both (1). Plasma membrane preparation has been reported from various cell types, BHK (4) and epithelial cells (16) but not from endothelial cells, with special focus on FGF receptors. The advantage of membrane preparation has already been well discussed (16).…”

“…Plasma membrane preparation has been reported from various cell types, BHK (4) and epithelial cells (16) but not from endothelial cells, with special focus on FGF receptors. The advantage of membrane preparation has already been well discussed (16).…”

“…The methods used to obtain such membrane fragments require certain modifications from the methods adopted for tissues both because of the fragility and low yield (1) as well as the in vitro conditions in which the cells are grown. Cell membranes have been isolated based on the differences in sedimentation and densities, as well as by chemical means (8,16). In this paper we describe a method that utilizes the correct conditions of sedimentation, density, osmotic pressure, and ionic strength for the preparation of membranes from coronary venular endothelial ceils (CVEC).…”