Figure 6.

Functional cell assays on self and self-like surfaces. (A) Adherent human microvascular endothelial cells (HMEC-1) opsonization assay. HMEC-1 were incubated with normal human serum (NHS) in the presence of increasing amounts (0.36–9.6 µM) of the different FH18-20 and FHR-1 analytes. The cells were labeled with anti-C3d antibody and an APC fluorescence marker to measure levels of C3-opsonization. The mean of the MFI of three independent assays (shown in Supplementary Figure 4 ), each conducted in duplicate, is plotted versus the protein concentration in µM with standard deviation (SD). As controls, NHS and heat inactivated serum (HIS) were used, each mixed with PBS. As a further control NHS was mixed with the highest concentration of glycine buffer used in the sample containing 9.6 µM of FHR-1(SV). MDFIs were calculated with the software tool FlowJo (version 7.6.5). (B) Sheep erythrocytes hemolysis assay. Sheep erythrocytes were incubated with NHS in the presence of increasing amounts (0.3–9.6 µM) of the different FH18-20 and FHR-1 analytes. Lysis and subsequent release of hemoglobin was determined by UV absorbance at 405 nm. All values were normalized to lysis in water (100% lysis mark). The mean percentage of hemolysis of three independent assays, each conducted in duplicate, is shown with SD and plotted against the protein concentration in µM. Controls were included as described for the HMEC-1 opsonization assay. A fourth control was added, which consisted of NHS mixed with PBS containing 5 mM EDTA. (C) PNH erythrocytes hemolysis assay. PNH erythrocytes were incubated with NHS in presence of increasing amounts (0.3–9.6 µM) of the different FH18-20 and FHR-1 analytes. Lysis was determined as described for the sheep erythrocytes hemolysis assay. The mean percentage of hemolysis of three independent assays, each conducted in duplicate, is shown with SD and plotted against the protein concentration in µM. The same controls as in the HMEC-1 opsonization assay were included.