High oxidative stress in SS cells persists across culture conditions with different O₂ concentrations. (A) uAA, uAS, and uSS cells were examined for CRR. The percentage of CRR⁺ cells (y-axis) was low in uAA and uAS cells and similar across all O₂ levels (P = .5021). As expected, uSS cells had a higher percentage positivity for CRR, almost fourfold higher than uAA (P < .0001). However, between O₂ conditions, the percentage of CRR⁺ cells in uSS did not change significantly (P = .6227). (B) A similar trend was seen in CRR⁺ cells normalized to parasitemia in iAA, iAS, and iSS cells. Although iAA and iAS cells had low positivity for CRR, iSS cells had 2.5-fold more CRR⁺ cells, as compared with iAA and iAS (P < .0001). This also indicates that most of the CRR positivity in iAA and iAS originates from parasite-harboring cells, because when normalized to parasitemia, they showed very few cells as CRR⁺. (C) Confocal images of iAA at 1%, 5%, 10%, and 21% O₂ levels and stained with CRR (red) and Vybrant DyeCycle (FITC). The images indicate complete colocalization between parasite (seen clearly in their classical 1N, 2N, or 4N forms) and CRR signal. Thus, ROS in iAA cells is mostly restricted to infected cells, and surrounding bystander cells have nondetectable levels of oxidative species. (D) Confocal images of iSS at 1%, 5%, 10%, and 21% O₂ levels and stained with CRR (red) and Vybrant DyeCycle (FITC). The images across different O₂ levels indicate presence of CRR signal in uninfected cells (shown in black arrows) and in addition, colocalization between parasite and CRR signal. Scale bar = 10 μm.