Large Particle Sorting to Isolate Live Parasitic Nematode Eggs

Alfonso Schmidt1, Tiffany Bouchery1, Graham Le Gros1, Kylie M. Price1

1 Malaghan Institute of Medical Research, Wellington
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.21
DOI:  10.1002/0471142956.cy1121s76
Online Posting Date:  April, 2016
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Abstract

Traditional jet‐in‐air cell sorters have been designed and optimized to isolate small particles such as mammalian lymphocytes with an average diameter of 10 μm. We discuss the practical considerations of setting up a conventional jet‐in‐air cell sorter, using a 200‐μm nozzle, to isolate the large parasitic nematode eggs of Nippostrongylus brasiliensis, with a maximum size of 60 μm. The eggs were separated based on light scattering properties, no fluorescent dye or molecule was required. © 2016 by John Wiley & Sons, Inc.

Keywords: large particle; cell sorting; live parasite; egg; nematode

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Harvesting N. brasiliensis Eggs from Rodent Feces for Cell Sorting
  • Support Protocol 1: Infection of Rodents and Collection of Parasite Worm
  • Basic Protocol 2: Purification of N. brasiliensis Eggs Using an Influx Cell Sorter with 200‐μm Nozzle
  • Support Protocol 2: Optimizing Drop Delay for 200‐μm Nozzle Using 30‐μm Beads
  • Support Protocol 3: Influx Sterilization Procedure
  • Support Protocol 4: Sterilization Quality Control
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Harvesting N. brasiliensis Eggs from Rodent Feces for Cell Sorting

  Materials
  • Feces from nematode infected rats or mice ( protocol 2)
  • Sterile distilled H 2O
  • Dulbecco's phosphate‐buffered saline (DPBS; Thermo Fisher Scientific, cat. no. 14190‐144) sterile
  • Sterile saturated NaCl salt solution (36 g/100 ml H 2O at 20°C; see recipe)
  • 50‐ml polypropylene tube, sterile
  • Vortex mixer
  • Cheesecloth cut in 7‐cm × 7‐cm squares
  • Centrifuge
  • 3‐ml transfer pipets
  • 25‐ml pipets
  • 70‐μm cell strainer, sterile
  • Glass slides
  • Stereomicroscope

Support Protocol 1: Infection of Rodents and Collection of Parasite Worm

  Materials
  • Infective third‐stage Nippostrongylus brasiliensis larvae (iL3)
  • Sterile Dulbecco's phosphate‐buffered saline (DPBS; Thermo Fisher Scientific, cat. no. 14190‐144)
  • 4‐month‐old female Lewis rats, or female Sprague‐Dawley rats with a minimum weight of 150 g, or mice with a minimum age of 7 weeks
  • 1‐ml disposable syringes, sterile
  • 50‐ml polypropylene tube, sterile
  • Stereomicroscope
  • 21‐ or 23‐G needles, sterile
  • Cage with wire‐grid floor (Techniplast)
  • Paper towels
  • Tablespoon
  • 25‐ml pipets
  • NOTE: Most strains of mice are susceptible to N. brasiliensis. We routinely use C57Bl/6 J mice.

Basic Protocol 2: Purification of N. brasiliensis Eggs Using an Influx Cell Sorter with 200‐μm Nozzle

  Materials
  • 10% bleach
  • 10× phosphate‐buffered saline (PBS, Thermo Fisher Scientific, cat. no. 70011‐044)), pH 7.4, sterile
  • Alignment beads (e.g., Ultra Rainbow Fluorescent Particles from Spherotech)
  • Accudrop beads (e.g., Accudrop Fluorescent Beads, BD FACS)
  • Cell sorter (equipped with a 488‐nm laser)
  • 200‐μm nozzle
  • 5‐ml tubes

Support Protocol 2: Optimizing Drop Delay for 200‐μm Nozzle Using 30‐μm Beads

  Additional Materials (also see protocol 3)
  • Alignment beads 30‐μm beads (e.g., Spherotech, cat. no. FP‐30052‐5)
  • Phosphate‐buffered saline (PBS; Thermo Fisher Scientific, cat. no. 70011‐044)
  • 5‐ml polypropylene tube
  • Microscope slide
  • Upright fluorescent microscope
  • 10× objective

Support Protocol 3: Influx Sterilization Procedure

  Materials
  • 70% (v/v) ethanol
  • dH 2O
  • 10% bleach
  • Sterile phosphate‐buffered saline (PBS; Thermo Fisher Scientific, cat. no. 70011‐044), 5 liters
  • Cell sorter

Support Protocol 4: Sterilization Quality Control

  Materials
  • Blood agar plate
  • Sterile cotton swabs
  • Cell sorter
  • Plastic bags (15 cm × 20 cm)
  • Incubator 37°C
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Figures

Videos

Literature Cited

Literature Cited
  Altman, P.L. and Katz, D.D. 1976. Cell biology. In Biological Handbooks, pp. 341‐346 FASEB, Bethesda, MD.
  Camberis, M., Le Gros, G., and Urban, J. Jr. 2003. Animal Model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Curr. Protoc. Immunol. 19.12.1‐19.12.27.
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  Stoeckius, M., Maaskola, J., Colombo, T., Rahn, H.P., Friedlander, M.R., Li, N., Chen, W., Piano, F., and Rajewsky, N. 2009. Large‐scale sorting of embryos reveals the dynamics of small RNA expression. Nat. Methods 6:745‐751. doi: 10.1038/nmeth.1370.
  Tylka, G.L., Niblack, T.L., Walk, T.C., Harkins, K.R., Barnett L., and Baker, N.K. 1993. Flow cytometric analysis and sorting of Heterodera glycines eggs. J. Nematol. 25:596‐602.
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Key References
  Arnold, L.W. and Lannigan J. 2010. Practical issues in high‐speed cell sorting. Curr. Protoc. Cytom. 51:1.24.1‐30. doi: 10.1002/0471142956.cy0124s51
  Camberis et al., 2003. See above.
  Galbraith, D.W. and Lucretti, S. 2000. Large particle sorting. In Flow Cytometry and Cell Sorting. 2nd ed. Springer, Berlin, Heidelberg.
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