Laminin-α5–adherent uRBCs are characterized by elevated intracellular Ca²⁺levels. (A) Intracellular Ca²⁺ levels of malaria culture–derived uRBCs (MC) normalized to control RBCs (control). Mean ± standard error of the mean (SEM); n = 6. (B) Intracellular Ca²⁺ levels of RBCs incubated in malaria conditioned medium (CM) normalized to control RBCs (control). Mean ± SEM; n = 7. (C) Intracellular Ca²⁺ levels of uRBCs derived from patients with malaria and age-matched control. Mean ± standard deviation (SD); n = 5-6; Mann-Whitney test. (D) Normalized laminin-α5 adherence of RBCs exposed to CM, with or without BAPTA treatment. Mean ± SEM, n = 4. (E) Mean fluorescence intensities (MFI) of Fluo-4 from malaria culture uRBCs (y-axis) were plotted against laminin-α5 adherence normalized to that of control RBCs (x-axis; n = 18). (F) Fluo-4–stained RBCs from control-, MC-, and CM-incubated RBCs were passed over laminin-α5–coated Ibidi μ-slides at a flow rate of 0.2 dyn/cm. Laminin-α5–bound RBCs were quantified and analyzed for Fluo-4 positivity by Arivis Vision 4D image analysis software. iRBCs were assessed by Hoechst staining. Mean ± SEM, n = 3-6. (G) uRBC ghosts were identified as spherical translucent Hoechst-negative cells and quantified over the course of 20 hours. Mean ± SEM; n = 4. (H) Ca²⁺ permeability of control RBCs (i), ionomycin-treated RBCs (ii), malaria cultured RBCs (iii), and RBCs incubated in malaria CM (iv) after 8 hours of experimental measurement. RBCs were stained for intracellular Ca²⁺ by Fluo-4 (green) and Hoechst (blue) for iRBC detection. (I) Representative microscopy images of RBCs incubated with iRBCs on laminin-α5–coated microscopic chambers at 0 hours of laminin-α5 incubation. Monocytes (arrows) were added after a 24-hour laminin-α5 incubation to carboxyfluorescein diacetate succinimidyl ester–stained RBCs (green), hemolyzed uRBCs (∗) and Hoechst-stained iRBCs (blue). (J) Phagocytosis of hemolyzed uRBCs (∗) by unstained monocytes (arrow) when co-incubated with iRBCs on laminin-α5–coated microscopic chambers. Red outlines show uRBC uptake by monocytes. Images in panels H, I, and J were obtained under a ×40 oil-immersion objective. Bars represent 20 μm. Wilcoxon matched-pairs signed-rank test, unless stated otherwise. Error bars denote SEM or SD.