Automated Synchronization of P. falciparum using a Temperature Cycling Incubator

Order of Publishing in Issue: 
Volume :5
Issue :2
April, 2011
Page No: 
Alejandro Almanza, [1, 2] Lorena Coronado, [1] Nicole Tayler, [1] Liuris Herrera [1, 2] and Carmenza Spadafora[1, 2]*
[1] Centro de Biología Celular y Molecular de las Enfermedades (CBCME), Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), Panama.
[2] International Cooperative Biodiversity Groups (ICBG), Smithsonian Tropical Research Institute (STRI), Panama.

Abstract : As malaria keeps affecting millions of lives every year, research based in culture of Plasmodium falciparum in vitro needs to beefficient and accurate. The development of better techniques and methodologies for the growth and maintenance of the parasites can save money,time, and lead to more trustable results. It has been observed, first in patients and then in the laboratory, that the malaria falciparum parasitesgrowth is affected by high temperatures. This trait can be used with laboratory cultures to synchronize and maintain the parasites in the samestage of their cell cycle. This harmony of stages is very desirable for the purpose of conducting metabolomic, proteomic and transcriptomeanalysis as well as for drug screening. Most scientists in the field of malaria use chemicals (usually sorbitol) that kill certain stages of theparasite to obtain synchronization, but this latter method does not last long and the parasites thus treated should not be used for assays immediatelyafter the treatment, due to the toxic effects that might have been infringed in the culture. A temperature cycling incubator (TCI) was acquiredin our laboratory and it was used to test the synchronization of the multidrug resistant W2 and chloroquine resistant 7G8 strains, commonly usedin our bioassays where they and their synchronization constitute essential tools for our drug discovery program. We followed the protocoldesigned by Haynes and Moch in 2002 and we made a comparison of the effectiveness of each of the two methods, chemical and temperaturebased. Our results show W2 synchronization by temperature cycling, with the help of an initial use of 0.3 M alanine, to last more than two monthswhile a tight synchronization with the use of 5% sorbitol was lost as rapidly as in one week. Sorbitol could also be used with the TCI forsynchronization with good results. However, 7G8 could not be efficiently synchronized with temperature cycling using the same program asthat of W2.

P. falciparum, Culture, Synchronization, High temperature, TCI.
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