Detection of Trypanosoma brucei variant surface glycoprotein switching by magnetic activated cell sorting and flow cytometry

Danae Schulz, Monica R. Mugnier, Catherine E. Boothroyd, F. Nina Papavasiliou

Research output: Contribution to journalArticlepeer-review

Abstract

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flowthrough contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Original languageEnglish (US)
Article numbere54715
JournalJournal of Visualized Experiments
Volume2016
Issue number116
DOIs
StatePublished - Oct 19 2016
Externally publishedYes

Keywords

  • African trypanosomiasis
  • Antigenic variation
  • Detection of trypanosoma brucei
  • Flow cytometry
  • Immunology
  • Issue 116
  • Switching
  • Variant surface glycoprotein

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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