Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles.
The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D006867:Hydrolases / Q000032:analysis; D007413:Intestinal Mucosa / Q000201:enzymology / Q000648:ultrastructure*; D007421:Intestine, Small / Q000201:enzymology / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008871:Microvilli / Q000201:enzymology / Q000648:ultrastructure*; D011817:Rabbits
D006867:Hydrolases