Abstract

BackgroundMolecular and genetic studies of blood-stage Plasmodium falciparum parasites require limiting dilution cloning and prolonged cultivation in microplates. The entire process is laborious and subject to errors due to inaccurate dilutions at the onset and failed detection of parasite growth in individual microplate wells.MethodsTo precisely control the number of parasites dispensed into each microplate well, parasitaemia and total cell counts were determined by flow cytometry using parasite cultures stained with ethidium bromide or SYBR Green I. Microplates were seeded with 0.2 or 0.3 infected cells/well and cultivated with fresh erythrocytes. The c-SNARF fluorescent pH indicator was then used to reliably detect parasite growth.ResultsFlow cytometry required less time than the traditional approach of estimating parasitaemia and cell numbers by microscopic examination. The resulting dilutions matched predictions from Poisson distribution calculations and yielded clonal lines. Addition of c-SNARF to media permitted rapid detection of parasite growth in microplate wells with high confidence.ConclusionThe combined use of flow cytometry for precise dilution and the c-SNARF method for detection of growth improves limiting dilution cloning of P. falciparum. This simple approach saves time, is scalable, and maximizes identification of desired parasite clones. It will facilitate DNA transfection studies and isolation of parasite clones from ex vivo blood samples.

Highlights

  • Molecular and genetic studies of blood-stage Plasmodium falciparum parasites require limiting dilution cloning and prolonged cultivation in microplates

  • The desired integration was confirmed by PCR prior to initiating limiting dilution cloning

  • Parasite dilution after flow cytometry data shows an improved hit rate To compare flow cytometry and different DNA staining methods in limiting dilution cloning experiments after transfection, the methods outlined in Fig. 1 were applied, establishing three experimental groups

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Summary

Introduction

Molecular and genetic studies of blood-stage Plasmodium falciparum parasites require limiting dilution cloning and prolonged cultivation in microplates. Two persisting and major bottlenecks, are the low transfection efficiencies in P. falciparum and the subsequent effort and time-consuming process of isolating clonal transfectant parasites. For confident characterization of single genotypes, limiting dilution cloning and prolonged cultivation in microplates is required and Macedo‐Silva et al Malar J (2021) 20:279 is the generally accepted standard [10]. This method is used for generating clones from ex vivo samples— e.g. clinical isolates or isolates from animals in genetic crosses

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