Purification Toxoplasma gondii Tissue Cysts Using Percoll Gradients

Elizabeth A. Watts1, Animesh Dhara2, Anthony P. Sinai2

1 Current Address: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, 2 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 20C.2
DOI:  10.1002/cpmc.30
Online Posting Date:  May, 2017
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Abstract

The protozoan parasite Toxoplasma gondii is capable of infecting all warm‐blooded animals and humans. Infectious, transmissible forms of the parasite include oocysts produced by the sexual cycle within the definitive feline host and tissue cysts that form Toxoplasma in the central nervous system and muscle during the asexual cycle within all chronically infected warm‐blooded hosts. These tissue cysts are populated with slow‐growing bradyzoites, which until recently have been thought to be dormant entities in the context of immune sufficiency. Reactivation to active growth during immune suppression is of critical clinical importance. However, little is known about tissue cysts or the bradyzoites they house, as the diversity of tissue cysts cannot be replicated in cell culture systems. This protocol for optimization of tissue cyst purification from the brains of infected mice using Percoll gradients provides an efficient means to recover in vivo–derived tissue cysts that can be applied to imaging, cell biological, biochemical, transcriptomic, and proteomic analyses. © 2017 by John Wiley & Sons, Inc.

Keywords: Toxoplasma gondii; tissue cyst; bradyzoite; Percoll

     
 
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Table of Contents

  • Introduction
  • Biosafety Considerations
  • Basic Protocol 1: Preparation of Infected Mouse Brain Homogenate
  • Basic Protocol 2: Establishing and Running Percoll Gradients
  • Basic Protocol 3: Imaging Purified Tissue Cysts
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Infected Mouse Brain Homogenate

  Materials
  • Spray bottle with 70% (v/v) ethanol
  • 10% (v/v) bleach
  • Sterile 1× phosphate‐buffered saline (PBS) with and without 1% (v/v) Tween‐80 (Sigma, cat. no. P4780)
  • Infected mice: e.g., CBA/J mice (The Jackson Laboratory, JAX000656) infected with Type II T. gondii strain ME49 (ATCC, 50611)
  • Dissection tools, sterilized in an autoclave pouch (Fig.  A,B):
    • Sharp fine‐point scissors, straight and curved
    • Round‐tip forceps
    • Stainless‐steel microspatula
  • Dissection surface (styrofoam lid covered with aluminum foil; Fig.  C)
  • 4‐liter plastic beaker
  • Biohazard bags for animal disposal
  • Large, low‐profile ice pan (e.g., Corning/Fisher, 432093)
  • Small porcelain mortar and pestle (e.g., Fisher, FB961B), sterilized in an autoclave pouch (Fig.  A,B)
  • 5‐ and 10‐ml pipettes
  • Sterile small‐head cell scraper (e.g., Corning, 3010)
  • 5‐ml Luer‐Lok syringes (Becton Dickerson, 309646)
  • Needles (Becton Dickerson):
    • 16‐G (purple; cat. no. 305198)
    • 18‐G (pink; cat. no. 305196)
    • 20‐G (yellow; cat. no. 305179)
    • 23‐G (green; cat. no. 305194)
  • Additional reagents and equipment for euthanasia
NOTE: When making 1× PBS/1% Tween‐80 from a 10× PBS stock, dilute the PBS first, then add Tween‐80 to a concentration of 1%.

Basic Protocol 2: Establishing and Running Percoll Gradients

  Materials
  • 10× PBS, filter‐sterilized
  • Percoll (Sigma, cat. no. P1644)
  • Sterile ddH 2O
  • Mouse brain homogenate (see protocol 1)
  • Color‐coded density calibration beads (Cospheric)
  • Sterile 50‐ml conical tubes (Falcon, 352070)
  • Sterile 14‐ml round‐bottom tubes (Falcon, 352059)
  • Sterile 5‐ml pipettes
  • Sterile 5‐ml syringes and 16‐G needles
  • Sealable BSL2‐certified centrifuge buckets
  • Tabletop centrifuge (Beckman Allegra 6R) with swinging bucket rotor (GH3.8) and 96‐well plate holder
  • Digital timer
  • Peristaltic pump (Rainin Dynamax)
  • 1.5‐ml microcentrifuge tubes and tube racks
  • Peristaltic pump tubing, 1.52 mm bore, 16 inch (Fisherbrand, 15‐190‐510)
  • Glass capillary tubes (SMI, model C, P5070‐902)
  • 96‐well flat‐bottom microtiter plate (Corning, 3596)
  • Inverted microscope with 10× or 20× objective

Basic Protocol 3: Imaging Purified Tissue Cysts

  Materials
  • Pellet of isolated tissue cysts (see protocol 2)
  • 1× phosphate‐buffered saline (PBS)
  • 70% (v/v) ethanol
  • Fixative (select one):
    • Absolute methanol, cold (−20°C)
    • 3% (w/v) paraformaldehyde (PFA; EMS, cat. no. RT15710) in PBS
  • 0.2% Triton X‐100 (Sigma, X100) in PBS
  • 10× Carbo‐Free Blocking Solution (Vector Laboratories, SP5040)
  • FITC‐conjugated Dolichos biflorus agglutinin (DBA; Vector Laboratories, FL‐1031)
  • Rhodamine‐conjugated concanavalin A (ConA; Vector Laboratories, FL‐1001)
  • 10 mg/ml Hoechst 33258 (Molecular Probes, H3569)
  • Mounting medium
  • Frosted glass slides (Thermo Fisher Scientific, 12‐544‐2)
  • Cytospin 4 Centrifuge (Thermo Scientific) with:
    • Cytospin funnels (Shandon TPX Sample Chamber, cat. no. A78710018)
    • Cytoclip Stainless‐Steel Slide Clips (Shandon, 59‐910‐052)
    • Cytospin filter cards (Fisherbrand, 22‐030‐410)
  • Beaker large enough to hold used clips/funnels
  • Coplin jars with screw‐on caps (Thermo Fisher Scientific, 08‐816)
  • Cardboard slide holder (Thermo Fisher Scientific, 12‐587‐10)
  • Wax pencil (Thermo Fisher Scientific, NC9072020)
  • Coverslips
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Figures

Videos

Literature Cited

Literature Cited
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