A rapid mechanical vibration procedure for purifying mature (Ia positive) columnar epithelial cells from the mouse small intestine.
A mechanical vibration method previously described for isolating gut epithelium was adapted to purify murine small intestinal epithelial cells in single cell suspension with high viability, good yield, optimal purity (99% epithelial cells with no other contaminating intestinal cells), and with preservation of phenotypical and physiological properties of epithelial cells. This rapid and easy procedure did not involve the use of any chelating agent or proteolytic enzyme. Extracted gut epithelial cells were suitable for in vitro functional assays within 45 min. Cell viability, as assessed by both the trypan blue due exclusion test and by titration of lactate dehydrogenase activity in the supernatant was greater than 75% up to 7 h after epithelial cell extraction. Light microscopy analysis of the duodeno-jejunal segment before and after mechanical vibration indicated that villous but not crypt epithelium was obtained using this method. Furthermore 99% of isolated epithelial cells expressed major histocompatibility class II (Ia) antigens indicating that the method was suitable for studies of the antigen presenting capacity of gut enterocytes.
Kaiserlian
D
D
Laboratoire d'Immunologie, INSERM U80 CNRS UA 1177 UCBL, Hôpital E. Herriot, Lyon, France.
Roche
C
C
Blanc
M
M
Revillard
J P
JP
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D004848:Epithelium / Q000648:ultrastructure; D005434:Flow Cytometry; D000949:Histocompatibility Antigens Class II / Q000032:analysis*; D007150:Immunohistochemistry; D007421:Intestine, Small / Q000276:immunology* / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D014732:Vibration
D000949:Histocompatibility Antigens Class II