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 (!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 scidb2m-/-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 TM 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. falciparuminfected 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. MALARIA is caused by the erythrocytic stages of protozoa of the genus Plasmodium, which colonize and destroy host's erythrocytes (1). To counter this disease, murine models of malaria are essential tools for research (2), particularly for drug discovery (3). In addition to the standard rodent experimental systems, different murine models of P. falciparum malaria are currently available (4-6). These are of special interest for drug discovery because, with the exception of human subjects, these are the only experimental systems available that allow the evaluation in vivo of the real human pathogen growing inside human erythrocytes (hE) previously engrafted into immunodeficient mice. Not surprisingly, the peripheral blood of these chimeric mice [humanized mice (HM)] is a complex mixture of murine erythrocytes (mE) and hE, in which the hematological effects of massive transfusions of hE and their elimination from peripheral blood may have important effects. Hence, the specific and quantitative measurement of different erythrocytic subpopulations is crucial in HM models, particularly when these models are used to establish the relationship between the amount of an antimalarial drug in blood and the effect on parasitemia through experimental pharmacokinetic and pharmacodynamic studies (PK/PD). In this kind of

Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model*

“…In the test groups, compound was administered twice daily on study days 4 -7, while animals in the Control group received no drug. Parasitemia was assessed by FACS as previously described (49).…”

IFN-γ–Induced Priming Maintains Long-Term Strain-Transcending Immunity against Blood-Stage Plasmodium chabaudi Malaria

“…with 5 3 10 8 mature iRBCs. Blood samples were collected at 1 h intervals and stained with SYTO16 (Molecular Probes, Life Technologies) as described previously (22 Parasite-specific ELISA PcAS-specific IgG1 and IgG2a serum levels were quantified by ELISA as described elsewhere (15). Briefly, 96-well flat-bottom microtest plates (Costar) were coated overnight (4˚C) with a total PcAS extract (10 mg/ml).…”

New approaches in antimalarial drug discovery and development: a review

“…The following nucleic acid dyes are used in P. falciparum tests: (i) SYBR Green I is the most widely used in vitro (Izumiyama et al 2009) and in vivo (Somsak et al 2012) and costs less than other dyes, (ii) YOYO-1 is the most sensitive dye and has the advantage of being able to detect low levels of parasitaemia, (iii) the SYTO series of dyes has increased applicability due to its binding affinities (Jiménez-Díaz et al 2009), (iv) propidium iodide, which is used with other stains, has a broad emission spectra that limits the range of dye combinations that can be used, (v) acridine orange, a relatively toxic molecule previously used for the diagnosis of human malaria, is laborious to use and has low reproducibility (Grimberg et al 2008), (vi) the Hoechst dye series, although selective, is of limited use particularly because of its high cost (Grimberg et al 2008) and (vii) PicoGreen provides reliable results, but is limited by the quenching of its fluorescence by haemoglobin, which confounds the detection of low levels of parasitaemia (Quashie et al 2006).…”