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Rapid isolation of single malaria parasite–infected red blood cells by cell sorting

Nature Protocols volume 6, pages 140–146 (2011)Cite this article

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Abstract

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum–infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.

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Figure 1: Purification of parasitized RBCs on a Percoll step gradient consisting of an upper 35% and a lower 65% Percoll layer.
Figure 2: Flow sorting of GFP-expressing cells.
Figure 3: Determination of singly infected RBCs from a typical scattergram.

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Acknowledgements

We thank the National Institute of Allergy and Infectious Diseases, National Institutes of Health (R01AI064553 and U19AI089672) for financial support.

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Authors and Affiliations

  1. Department of Entomology, The Pennsylvania State University, University Park, Pennsylvania, USA

    Jun Miao & Liwang Cui

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  1. Jun Miao
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  2. Liwang Cui
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Contributions

J.M. designed and performed the experiments, analyzed the data and wrote the paper; L.C. supervised the project and wrote the paper.

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Correspondence to Jun Miao or Liwang Cui.

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The authors declare no competing financial interests.

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Miao, J., Cui, L. Rapid isolation of single malaria parasite–infected red blood cells by cell sorting. Nat Protoc 6, 140–146 (2011). https://doi.org/10.1038/nprot.2010.185

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