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 gels 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.
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Venkateswaran, V., Hosick, H.L. Assay of ligand receptors on membranes of epithelial cells cultured in three dimensional matrices. Journal of Tissue Culture Methods 13, 261–263 (1991). https://doi.org/10.1007/BF02388259
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DOI: https://doi.org/10.1007/BF02388259