title: Synchronous culture of Plasmodium falciparum at high parasitemia levels.
abstract: This protocol describes a method for preparing cultures of Plasmodium falciparum synchronized at any intraerythrocytic stage. Using this method, around 60% parasitized cells may be obtained. On the basis of Trager and Jensen's original continuous culture method, our approach relies on the use of fresh human blood not older than 2 weeks, a low hematocrit between 0.8 and 1.5%, a starting frozen inoculum of 10% ring-stage parasitemia, human serum replaced with AlbuMAX I and alternating sorbitol and Percoll synchronization methods to shorten the cycle window to 4-6 h and reduce sorbitol toxicity. From our synchronized high parasite density cultures, 3-5 ml of infected red blood cells can be obtained in 1 week, corresponding to 1.2 mg of total parasite protein per ml of harvested culture. On the basis of the variables parasitemia and packed cell volume, we provide an equation to accurately calculate the amount of complete medium required every 24 h corrected for the cycle stage and capacity of the culture flask. Ten days suffice to complete the protocol from a frozen stock of parasites.
authors.affiliation: Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Ciudad Universitaria, Madrid 28040, Spain.
authors.forename: Azar
authors.initials: A
authors.lastname: Radfar
authors.forename: Darío
authors.initials: D
authors.lastname: Méndez
authors.forename: Carlos
authors.initials: C
authors.lastname: Moneriz
authors.forename: María
authors.initials: M
authors.lastname: Linares
authors.forename: Patricia
authors.initials: P
authors.lastname: Marín-García
authors.forename: Antonio
authors.initials: A
authors.lastname: Puyet
authors.forename: Amalia
authors.initials: A
authors.lastname: Diez
authors.forename: José M
authors.initials: JM
authors.lastname: Bautista
mesh_terms: D018929:Cell Culture Techniques; D003470:Culture Media; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D010963:Plasmodium falciparum / Q000254:growth & development* / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism
chemical_list: D003470:Culture Media; D015800:Protozoan Proteins