Isolation of Primary Cilia by Shear Force

Kimberly A. P. Mitchell1

1 Department of Biology, Liberty University, Lynchburg, Virginia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.42
DOI:  10.1002/0471143030.cb0342s59
Online Posting Date:  June, 2013
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Abstract

The cell's primary cilium is both a mechanical and chemical sensor involved in many signaling pathways. In order to ascertain protein enrichment in the primary cilium or study sub‐ciliary localization of various proteins, it is advantageous to remove the primary cilium from the cell body. The protocol described here gives detailed instructions on purifying primary cilia by separating them from the cell body using shear force. This simple technique avoids using harsh purification conditions that may affect signaling proteins in the cilium or cause the ciliary membrane to disintegrate. In addition, as the cell body remains mostly intact, contamination of the isolated cilia by proteins from the cell body is minimized. This protocol is ideally suited for isolating cilia from renal cell lines, as primary cilia in these cells grow to greater lengths than in other cell types (up to 50‐µm long in Xenopus A6 toad kidney cells as opposed to 1 to 5 µm in NIH3T3 fibroblast cells). Curr. Protoc. Cell Biol. 59:3.42.1‐3.42.9. © 2013 by John Wiley & Sons, Inc.

Keywords: primary cilia; isolation; purification; shear force

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolating Primary Cilia Using Shear Force
  • Basic Protocol 2: Visualization of Isolated Primary Cilia Fraction Using Electron Microscopy (EM)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolating Primary Cilia Using Shear Force

  Materials
  • Cells such as Xenopus A6 cells
  • A6 tissue culture medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Resuspension buffer (see recipe)
  • 150‐mm tissue culture dishes
  • Humidified incubator with CO 2 supply
  • Vacuum aspirator
  • Rotary shaker
  • ∼50‐ml centrifuge tubes appropriate for chosen rotor
  • Micropipettor
  • Refrigerated centrifuge (Beckman J6 or equivalent) with either swinging‐bucket or fixed‐angle rotor capable of volumes of 45 ml or more
  • Felt tip pen
  • ∼24‐ml ultracentrifuge tubes appropriate for chosen rotor
  • Refrigerated ultracentrifuge with fixed‐angle rotor (Beckman 60Ti, or equivalent)

Basic Protocol 2: Visualization of Isolated Primary Cilia Fraction Using Electron Microscopy (EM)

  Materials
  • Isolated primary cilia (see protocol 1)
  • Resuspension buffer (see recipe)
  • 2% uranyl acetate solution
  • Parafilm
  • Formvar‐coated carbon grids (200‐mesh; Ted Pella)
  • Fine forceps
  • Filter paper
  • Electron microscope such as Phillips CM12 or equivalent
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Figures

Videos

Literature Cited

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