Isolation of ciliated or unciliated basal bodies from the rabbit oviduct

Abstract

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 KCI, 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 j/z 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 CaC12, 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. © 1974, Rockefeller University Press., All rights reserved.