Techniques have been developed for the isolation of basal bodies with cilia attached or for the isolation of only basal bodies from the rabbit oviduct. Oviducts are removed, cut open, and placed in an extraction medium composed of 0.25 M sucrose, 0.001 M EDTA, 0.025 M KCl, 0.02 M Hepes buffer pH 7.5, and 0.05% Triton X-100. After the oviduct is agitated in this medium on a Vortex mixer for ½ h, the lumenal cortex of each ciliated cell, containing 200–300 basal bodies with cilia attached, is released as a unit. The cortices and the intact nuclei, which are also released from the disrupted cells, form a pellet when the extraction medium is centrifuged at 600 g for 10 min. When cortices which contain only basal bodies are to be isolated, the oviduct is subjected to conditions which remove the cilia prior to being processed as above. The cilia are removed when the oviduct is placed in a medium of 0.25 M sucrose, 0.01 M CaCl2, 0.02 M Pipes buffer pH 5.5, and 0.05% Triton X-100 and continuously agitated for 15 min on a Vortex mixer. The low pH and Ca++ solubilize the transition region of the cilium and also prevent the cell from being disrupted. The cortices can be partially purified if the 600-g pellet is resuspended in 2.2 M sucrose pH 6.5 and centrifuged at 40,000 g for 2 h. Under these conditions, 85% of the nuclei form a pellet and the cortices float to the surface of the sucrose. In addition to the basal bodies or basal bodies with cilia, the cortices contain some adherent cytoplasm, a few fibers, and a few vesicles which may be remnants of mitochondria or endoplasmic reticulum. The structure of the cilia and the basal bodies isolated with either procedure is normal.
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Article| February 01 1974
ISOLATION OF CILIATED OR UNCILIATED BASAL BODIES FROM THE RABBIT OVIDUCT
Richard G. W. Anderson
From the Oregon Regional Primate Research Center, Beaverton, Oregon 97005.
Dr. Anderson's present address is the Department of Cell Biology, University of Texas Southwestern Medical School, Dallas, Texas 75235.
Received: July 27 1973
Revision Received: October 18 1973
Online Issn: 1540-8140
Print Issn: 0021-9525
Copyright © 1974 by The Rockefeller University Press
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Richard G. W. Anderson; ISOLATION OF CILIATED OR UNCILIATED BASAL BODIES FROM THE RABBIT OVIDUCT . J Cell Biol 1 February 1974; 60 (2): 393–404. doi: https://doi.org/10.1083/jcb.60.2.393
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