Recombinant laminin 511

RBC adhesion to laminin-α5 and HA was assessed by applying 0.5 μg recombinant laminin-511 (BioLamina, Sundyberg, Sweden) or 2.5 mg/mL HA (Sigma-Aldrich) diluted in HEPES buffer (132 mM NaCl, 20 mM HEPES, 6 mM KCl, 1 mM MgSO₄, 1.2 mM K₂HPO₄, and 1 mM Ca²⁺) through passive absorption on uncoated Ibidi μ-slides^0.4 or ibiTreat μ-slides^0.4 in flow chambers (Ibidi, Gräfelfing, Germany). Adhesion and rolling frequency were visualized by EVOS microscopy (ThermoFisher, Waltham, MA) and LSM 510 META/TIRF (Carl Zeiss Microimaging, Jena, Germany) and analyzed by Vision4D (Arivis, Rostock, Germany) at a flow rate of 0.2 dyn/cm. The blocking of CD44 and HA interaction was mediated via 18 μg/mL anti-CD44 antibody (Invitrogen, Carlsbad, CA). Anti-hLu/BCAM (2 μg/mL) blocked Lu/BCAM (R&D Systems, Minneapolis, MN) and laminin-α5 interaction. CM RBCs were treated with 25 μM BAPTA-AM (Sigma-Aldrich) overnight, to test the effect of Ca²⁺ chelation on laminin-α5 adherence to uRBCs. Heat inactivation of the malaria-conditioned medium at 56°C for 1 hour was performed to further characterize the altering of the adhesion capacity of the uRBCs by the parasite-derived soluble factor.

Fluo-4-stained RBCs (0.5 × 10⁶; 1 μM; Invitrogen) were added to microscopic chambers (Nunc Laboratory-Tek Chambered Coverglass; ThermoFisher Scientific, Waltham, MA) coated with 0.5 μg recombinant laminin-511 (BioLamina, Sundyberg, Sweden) for 2 hours at 37°C. IRBCs were stained with 1 μg/mL Hoechst (Sigma, Spruce). Ionomycin (Sigma-Aldrich) treated RBCs (5 μM for 30 minutes at 37°C) were used as a positive control for increased intracellular Ca²⁺ levels. Ca²⁺ permeability was visualized with Observer Z1 (Zeiss, Oberkochen, Germany) taking frames of a defined area in the microscopic chamber every 5 minutes for 21 hours. Images were analyzed by ZEN blue image analysis software (Zeiss) to determine mean fluorescence intensities for Fluo-4 and for the quantification of translucent spherical Hoechst-negative uRBC ghosts.