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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Brian Lyko, Elizabeth A Hammershaimb, Wang Nguitragool, Thomas E Wellems, Sanjay A Desai
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-124) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

BL, EAH, and WN designed and performed experiments and carried out data analysis. TEW provided reagents and guidance on limiting dilution. SAD conceived the project, analysed data, and wrote the paper. All authors read, revised and approved the manuscript.

Abstract

Background

Molecular and cellular studies of Plasmodium falciparum require cloning of parasites by limiting dilution cultivation, typically performed in microplates. The parasite's slow replication rate combined with laborious methods for identification of positive wells has limited these studies. A new high-throughput method for detecting growth without compromising parasite viability is reported.

Methods

In vitro parasite cultivation is associated with extracellular acidification. A survey of fluorescent pH indicators identified 5-(and-6)-carboxy SNARF-1 as a membrane-impermeant dye with a suitable pK a value. Conditions for facile detection of viable parasites in 96-well microplates were optimized and used for limiting dilution cloning of genetic cross progeny and transfected parasites.

Results

5-(and-6)-carboxy SNARF-1 is a two-emission wavelength dye that accurately reported extracellular pH in parasite cultures. It readily detected parasite growth in microplate wells and yielded results comparable to labour-intensive examination of Giemsa-stained smears. The dye is non-toxic, allowing parasite detection without transfer of culture material to additional plates for separate assays. This dye was used with high-throughput limiting dilution culture to generate additional progeny clones from the HB3 × Dd2 genetic cross.

Conclusions

This fluorescence-based assay represents a low-cost, efficient method for detection of viable parasites in microplate wells; it can be easily expanded by automation.
Zusatzmaterial
Authors’ original file for figure 1
12936_2011_2072_MOESM1_ESM.pdf
Authors’ original file for figure 2
12936_2011_2072_MOESM2_ESM.pdf
Authors’ original file for figure 3
12936_2011_2072_MOESM3_ESM.pdf
Literatur
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