Isolation of primary cilia for morphological analysis.
Primary cilia are present in most mammalian cells and have lately been recognized as important cellular sensors that integrate and transduce extracellular signals into functional responses. Development of approaches to isolate primary cilia of sufficient quantity and quality for biochemical and molecular studies are crucial to understand their roles and functions under normal and pathological conditions. Two separate but complementary techniques (i.e., peel-off and slide pulling) to isolate enriched ciliary fractions from cultured epithelial cells are described. The purity and quantity of isolated cilia is verified by immunofluorescent confocal microscopy, light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and western blot analysis. Examples of detection of ciliary-associated proteins using isolated cilia are shown. These techniques will allow the isolation of primary cilia from cultured epithelial cells and permit further examination of the expression and localization of proteins of interest, helping to elucidate the role of primary cilia in health and disease.
Huang
Bing
B
Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Masyuk
Tatyana
T
LaRusso
Nicholas
N
D000818:Animals; D018929:Cell Culture Techniques; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods; D002460:Cell Line; D002923:Cilia / Q000737:chemistry* / Q000648:ultrastructure; D010179:Pancreas