Shedding of tumor cell surface membranes

Mastocytoma P815 cells are induced to form and shed membrane vesicles (MV) from their surfaces by incubation at low temperature (4 degrees C) for 1 hr. and subsequently allowing them to warm up to room temperature (22 degrees C). Within 1-2 hrs. at room temperature, up to 90% of the P815 cells form and shed MV from their surfaces. Both cells and vesicles remain trypan blue-excluding during the MV shedding process. This process is energy dependent in that it can be inhibited by 2-deoxyglucose, sodium azide and 2-4-dinitrophenol. The shed MV can be harvested by centrifugation on a 6% Ficoll cushion and quantitated in terms of protein content. The shedding of membrane vesicles from the tumor cell surfaces results in a significant reduction in the cell size.