Extended Data Fig. 5. Gating strategy for detection of erythrocyte binding Ca²⁺ uptake by flow cytometry.

a. Example scatter plots of the gating strategy for the erythrocyte binding assay showing unstained erythrocytes and erythrocytes incubated with PCRCR and detected with anti-PfRipr polyclonal sera and Alexa-488. The erythrocyte population was gated with SSC-A and FSC-A (top), then doublets were excluded using FSC-H and FSC-A (middle). For determining complex binding to erythrocytes, a cutoff of >10³ was used (bottom). Gating was performed in an identical manner for all other antibody and antigen combinations. b. Kinetic plot of A23187 stimulation of erythrocytes pre-loaded with Fluo-4 AM. A titration of A23187 shows the responsiveness of the Fluo-4 AM labelled erythrocytes to the calcium ionophore (top). The mean fluorescence intensity for the Fluo-4 AM signal is plotted. Kinetic plot of PCRCR addition to erythrocytes in comparison to 0.5 μM A23187 stimulation (bottom).