title: Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.
abstract: Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (> or =0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD(scidbeta2m-/-) engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparum-infected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation.
authors.affiliation: GlaxoSmithKline R & D, ID CEDD Diseases of the Developing World, Tres Cantos, Spain.
authors.forename: María Belén
authors.initials: MB
authors.lastname: Jiménez-Díaz
authors.forename: Teresa
authors.initials: T
authors.lastname: Mulet
authors.forename: Vanesa
authors.initials: V
authors.lastname: Gómez
authors.forename: Sara
authors.initials: S
authors.lastname: Viera
authors.forename: Angela
authors.initials: A
authors.lastname: Alvarez
authors.forename: Helena
authors.initials: H
authors.lastname: Garuti
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Vázquez
authors.forename: Alejandra
authors.initials: A
authors.lastname: Fernández
authors.forename: Javier
authors.initials: J
authors.lastname: Ibáñez
authors.forename: Magdalena
authors.initials: M
authors.lastname: Jiménez
authors.forename: Domingo
authors.initials: D
authors.lastname: Gargallo-Viola
authors.forename: Iñigo
authors.initials: I
authors.lastname: Angulo-Barturen
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004912:Erythrocytes / Q000166:cytology / Q000469:parasitology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008288:Malaria / Q000469:parasitology*; D051379:Mice; D008815:Mice, Inbred Strains; D016513:Mice, SCID; D010961:Plasmodium / Q000302:isolation & purification*
chemical_list: D005456:Fluorescent Dyes