An automated method for determining the cytoadhesion of ...
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An automated method for determining the cytoadhesion of ...
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Hempel et al. Malaria Journal (2015) 14:112 DOI 10.1186/s12936-015-0632-4
METHODOLOGY
Open Access
An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells Casper Hempel1*, Ida M Boisen1,2, Akinwale Efunshile3,4, Jørgen AL Kurtzhals1,2 and Trine Staalsø2
Abstract Background: Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin. Methods: Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots. Results: The manual and automated quantification showed strong, positive correlation (r2 = 0.959, p
