Isolation of plasma membranes from rat C6 glioma cells cultivated on microcarriers

We have studied the feasibility of rat C6 glioma cell cultivation on microc arrier beads and the isolation of their plasma membranes from the beads. Ce lls were cultivated on Cytodex-1 microcarrier beads and the plasma membrane s were subsequently isolated from confluent cell monolayers on the beads. T his approach yielded approximately 4 x 10(6) cells/ml in a 1 L spinner vess el. Enzymatic assays indicated an 18-fold enrichment of plasma membranes is olated from the beads with minor contamination by other cell organelles. As say for IGF-I receptor binding capacity revealed that 70% of the total rece ptor binding capacity could be recovered in the plasma membrane fraction is olated from the beads as compared with the receptor binding capacity of int act cells, demonstrating the functional integrity of the isolated membranes . Electron microscopy and immunofluorescence analysis indicated that the is olated plasma plasmic surface. Our procedure of C6 glioma cell cultivation on microcarriers and subsequent plasma membrane isolation, provides large q uantities of homogenous and metabolically active membranes which can be use d to study receptor-mediated effects on cell proliferation and differentiat ion.