Redox imbalance in host SS cells may provide a less hospitable environment for Babesia: B divergens was cultured under standard culture conditions in AA, AS, and SS cells. Redox assays were performed on day 2 after the invasion. (A) Using a cell-permeant and cytoplasm-specific redox dye CRR, we quantified the percentage of cells with ROS. Left panel: uAA and uAS had negligible levels of ROS, whereas that of uSS was almost sixfold higher (P = .0018, n = 11). Right panel: different genotypes exhibit differential parasitemia (Pt%); thus, for infected cells, we normalized CRR signal to Pt%; iSS signal was threefold higher than iAA and iAS (P = .028, n = 15). (B) Cells were stained with HO (DNA dye which is equivalent to Pt% in a nucleus-free RBC) and CRR. Cells positive for CRR were used as parent gate and checked for HO positivity. For iAA, iAS, and iSS, most CRR⁺ cells were HO⁺, whereas only in uSS samples, they showed CRR signal. This was quantified in the graph where y-axis represents the percentage of HO⁺ cells in the CRR⁺ population in each of the genotypes. As shown, in AA and AS cells, there was almost complete coincidence of oxidative stress in infected (HO⁺) cells, whereas in SS cells, only 42.7% ± 4.1% CRR⁺ cells harbored parasites (P < .0001). (C) Cells were stained with CRR (red) and Vybrant DyeCycle (fluorescein isothiocyanate [FITC]) DNA dye, and confocal microscopy was performed using 63× magnification using Zeiss LSM 880 with Airyscan. As shown in the image, uAA had no CRR signal, but several uSS showed positivity for CRR (marked with black arrows). In iAA, the ROS signal coincided completely with cells harboring the parasite, whereas in iSS, uninfected bystander cells also showed fluorescence (marked in black arrows). Thus, imaging of these cells strengthens our findings and provides a visual picture of our flow data. (D) Cells were stained with DCFDA, a redox dye which detects hydroxyl species, peroxyl species, and other ROS species within cells, detected in the 488-D channel in BD Fortessa flow cytometer. As shown in histogram overlays, both uSS and iSS (orange, pink, and green shading) were right shifted compared with uAA and iAA (in blue), respectively. Scale bars = 10 μm.