title: Purification and in vitro selection of rosette-positive (R+) and rosette-negative (R-) phenotypes of knob-positive Plasmodium falciparum parasites.
abstract: We have developed methods for in vitro selection of Plasmodium falciparum parasites that bear knob protrusions (K+) and are either of the rosette-positive (K+R+) or rosette-negative (K+R-) phenotypes. Cryopreserved parasites from spleen-intact Aotus monkeys that were K+, C32 cell adherence-positive (C+), CD36 adherence-positive, and R- with Aotus erythrocytes were adapted to continuous growth in human erythrocytes, and selected initially for adherence to C32 melanoma cells. In the absence of independent selection for rosettes, K+R-C+ parasites were produced that adhered to both C32 cells and CD36. Without selection for the C+ phenotype, K+R-C- parasites eventually predominated in such cultures. The R+ parasites were selected using differences in sedimentation behavior of rosette-infected cells versus non-rosette-infected cells. Methods were devised for selection of the R+ or R- phenotypes and for the purification of R+ or R- infected cells of high parasitemia that were suitable for molecular studies. With the repeated selection for K+R+ parasites, we were able to maintain the K+R+ phenotype for several months in vitro. These methods will allow systematic study of the molecular basis of the K+R+ and K+R- phenotypes.
authors.affiliation: Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California.
authors.forename: S M
authors.initials: SM
authors.lastname: Handunnetti
authors.forename: A D
authors.initials: AD
authors.lastname: Gilladoga
authors.forename: M R
authors.initials: MR
authors.lastname: van Schravendijk
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: M
authors.initials: M
authors.lastname: Aikawa
authors.forename: R J
authors.initials: RJ
authors.lastname: Howard
mesh_terms: D000818:Animals; D001029:Aotus trivirgatus; D002448:Cell Adhesion; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D008854:Microscopy, Electron; D010641:Phenotype; D010963:Plasmodium falciparum / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure; D012397:Rosette Formation