Primary neutrophils or differentiated PLB985 cells were incubated with fluorescent late‐stage iRBC either expressing GFP or stained using MitoTracker as described, at a 10:1 ratio at 37°C for different time periods. Samples were washed, and the extent of neutrophils–iRBC interaction (% fluorescent neutrophils) was determined using flow cytometry. Opsonization of iRBCs was performed by culturing iRBCs with AB human serum (Sigma) for 30 min at 37°C. To assess ligand–receptor specificity to this interaction, we performed these assays using anti‐Cd11b antibody (Biolegend Cat # 101211, 10 μl/ml) and a non‐PfEMP1‐blocking anti‐ICAM‐1 antibody (Biolegend Cat # 322702, 10 μl/ml) as negative controls. An anti‐ICAM‐1 monoclonal antibody (15.2) that blocks the PfEMP1‐binding site (Thermofisher, MA180910, 10 μl/ml) was used as blocking antibody as described (Baratin et al, 2007 (link)). All antibodies were incubated with iRBCs for 30 min at room temperature in culture media prior to flow cytometry interaction assays. To inhibit phagocytosis, neutrophils were pre‐incubated with 5 µM cytochalasin D (Sigma‐Aldrich), an actin polymerization inhibitor, for 1 h at 37°C prior to the addition of fluorescent iRBCs.