AN IMPROVED APPROACH TO FREEZE-FRACTURE MORPHOLOGY OF MONOLAYER CELL-CULTURES

Much work is currently done on cell cultures to elucidate membrane pro cesses associated with different cell functions. We describe here a mo dified freeze-fracture method to obtain systematically large fractured areas of the plasma membrane from monolayer cell culture in situ. Cel ls are grown until confluence on a Thermanox(C) coverslip overlaid wit h poly-L-ornithine. After chemical fixation, the culture is flattened overnight by sandwiching it between the Thermanox(R) coverslip, a Falc on(R) membrane and a glass coverslip, under a 5 g weight. After freeze -fracture, vast pictures of the protoplasmic leaflets are obtained in a reproducible manner. Our approach was applied to cultures which were stimulated to release acetylcholine; it has been found very appropria te for studying modifications affecting intramembrane particles and ve sicles openings in the plasmalemma. Accurate quantifications were perf ormed and correlations were established between the membrane changes a nd the data revealed by thin sections. The present sandwich method can be applied to a variety of cell preparations, allowing for quantitati ve study of structure-function relationships. (C) 1998 Elsevier Scienc e B.V. All rights reserved.