An alternative protocol for Plasmodium falciparum culture synchronization and a new method for synchrony confirmation

Malaria Journal

Methodology

Channe Gowda¹  Ronnie A Childs²  Liwang Cui²  Jun Miao² 

[1] Department of Biochemistry and Molecular Biology, Pennsylvania State University, College of Medicine, PA, 17033, Hershey, USA;Department of Entomology, Pennsylvania State University, University Park, PA, 16802, USA;

关键词: SyBr Green I;    Plasmodium falciparum;    Egress;    Invasion;    Culture synchronization;   

DOI  :  10.1186/1475-2875-12-386

 received in 2013-07-04, accepted in 2013-10-22,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundAlthough the Percoll/sorbitol synchronization method is widely accepted, its use for achieving tight synchronizations is cumbersome. In addition, subsequent conclusions on the synchrony status are often based on visual inspection of parasites and few reports provide an unbiased estimate confirming the degree of synchrony. This report presents a simpler synchronization procedure and offers an objective method to validate parasite synchrony.MethodsParasite synchronization was performed by culturing late-stage schizont parasites for a defined period of time, subjecting them to Percoll density centrifugations, and collecting the newly formed rings. Repeating the process several times on the un-egressed schizonts maximizes the recovery of several synchronized ring-stage parasite populations. The culture synchrony for each population was verified by allowing the synchronized rings to mature to late-stage schizonts and collecting ring-stage sample aliquots at three-hour intervals for nine hours. The aliquots were then measured, using the SyBr Green I assay, to determine when the ring-stage parasitaemia stops increasing.ResultsQuantitative measurements of ring-stage parasites showed that under the conditions described, a four to six-hour synchrony period is obtained.ConclusionBy taking advantage of Plasmodium’s periodic lifecycle in erythrocytes, it is shown that Percoll density centrifugation alone is sufficient to tightly synchronize cultures with minimal parasite loss. In addition, the degree of culture synchrony is validated using the SyBr Green I assay.

【 授权许可】

CC BY   
© Childs et al.; licensee BioMed Central Ltd. 2013