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.