Synchronous culture of Plasmodium falciparum at high parasitemia levels.
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.
Radfar
Azar
A
Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Ciudad Universitaria, Madrid 28040, Spain.
Méndez
Darío
D
Moneriz
Carlos
C
Linares
María
M
Marín-García
Patricia
P
Puyet
Antonio
A
Diez
Amalia
A
Bautista
José M
JM
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
D003470:Culture Media; D015800:Protozoan Proteins