Figure 3. Flow cytometric analyses of parasitized erythroblasts.

C57BL/6 mice were infected with GFP-expressing malaria parasites. (a) Peripheral blood or spleen cells were stained with PE-Cy7 conjugated anti-TER119 Ab on day 8 and 18. Spleen cells were treated with or without lysis buffer before staining. Data are from one representative of at least four independent experiments. (b) The percentages of the GFP⁺ cells within TER119⁺ cells in the peripheral blood (parasitemia, open circles) and those in spleen cells after lysis (infected erythroblasts, closed circles) were calculated (upper panel). Data are mean ± SD from six mice. Hematocrits were evaluated to monitor anemia in mice infected with PyNL (lower panel). Data are ± SD from 10 mice. (c) Cells from the indicated organs on day 20 were stained with PE-Cy7 conjugated anti-TER119 Ab and PE-conjugated anti-CD44 Ab, or anti-MHC class I Ab. GFP expression in erythroblasts in mice infected with GFP-PyNL and the gating strategies (top panels). First, live cells were gated by forward scatter (FSC) and side scatter (SSC), then erythroid cells were gated as TER119⁺ cells expression, and infected erythroid cells were gated as GFP⁺ cells. A histogram shows GFP expression of erythrocytes (peripheral blood) and erythroblasts (lysed spleen cells) infected with GFP-PyNL. Cells after gating by FSC and SSC were analyzed for TER119 and GFP as in (a). Eythroid cells were analyzed for CD44, MHC class I, isotype control for MHC class I in right 3 columns. Numbers indicate percentage of boxed cells in gated population (not total cells). (d) Infected and uninfected erythroid cells were analyzed for expression of MHC class I. Data comprise one representative of three independent experiments.