Isolation of plasma membranes from rat C6 glioma cells cultivated on microcarriers.
We have studied the feasibility of rat C6 glioma cell cultivation on microcarrier beads and the isolation of their plasma membranes from the beads. Cells were cultivated on Cytodex-1 microcarrier beads and the plasma membranes were subsequently isolated from confluent cell monolayers on the beads. This approach yielded approximately 4 x 10(6) cells/ml in a 1 L spinner vessel. Enzymatic assays indicated an 18-fold enrichment of plasma membranes isolated from the beads with minor contamination by other cell organelles. Assay for IGF-I receptor binding capacity revealed that 70% of the total receptor binding capacity could be recovered in the plasma membrane fraction isolated from the beads as compared with the receptor binding capacity of intact cells, demonstrating the functional integrity of the isolated membranes. Electron microscopy and immunofluorescence analysis indicated that the isolated plasma membranes were highly homogeneous with the majority exposing the cytoplasmic surface. Our procedure of C6 glioma cell cultivation on microcarriers and subsequent plasma membrane isolation, provides large quantities of homogeneous and metabolically active membranes which can be used to study receptor-mediated effects on cell proliferation and differentiation.
Wang
Q
Q
Department of Biochemistry, University of Antwerp, Belgium.
Slegers
H
H
Clauwaert
J
J
D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002470:Cell Survival; D003911:Dextrans / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005910:Glioma / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003911:Dextrans; C052839:Cytodex; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D017526:Receptor, IGF Type 1