Protocol | Published:

An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development

Nature Protocols volume 10, pages 11311142 (2015) | Download Citation

Abstract

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.

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Acknowledgements

We thank D. Ravel for critical reading of the manuscript. This work has been supported through a career development grant from the Burroughs Wellcome Fund and by US National Institutes of Health (NIH) grants R21 AI105328 and RC1AI086222 (M.M.). N.M.B.B. is supported by a postdoctoral fellowship from the Swiss National Science Foundation and K.B. was supported through a Feodor Lynen postdoctoral fellowship from the Alexander von Humboldt Foundation.

Author information

Author notes

    • Kathrin Buchholz

    Present address: Biochemistry and Molecular Biology, Justus Liebig University Giessen, Giessen, Germany.

    • Nicolas M B Brancucci
    •  & Ilana Goldowitz

    These authors jointly directed this work.

Affiliations

  1. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

    • Nicolas M B Brancucci
    • , Ilana Goldowitz
    • , Kathrin Buchholz
    • , Kristine Werling
    •  & Matthias Marti

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Contributions

I.G., N.M.B.B. and M.M. wrote the manuscript, with input from all coauthors. K.B. and M.M. conceived and developed the originally published protocol. I.G. conceived and performed preliminary experiments and N.M.B.B. conceived and performed later experiments leading to improvements in the protocol. N.M.B.B. and K.W. generated the data shown in the figures. All authors revised the manuscript and contributed to interpretation of the results.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthias Marti.

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DOI

https://doi.org/10.1038/nprot.2015.072

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