Figure 3. Downstream functional analysis of amplification pathways 1 and 2 by C4 cleavage assay and phagocytosis assay.

(A) C4 cleavage assay with PC and GlcNAc as ligands in normal (pH 7.4, 2.5 mM calcium) and infection-inflammation (pH 6.5, 2 mM calcium) conditions. PC beads were incubated with amplification pathway 1 components (CRP, L-ficolin, MASP-2, or C4) and GlcNAc beads were incubated with amplification pathway 2 components (L-ficolin, CRP, C1, or C4) under normal and infection-inflammation conditions. The cleaved C4 deposited on the beads were detected by Western blot. Negative controls were beads incubated with complete amplification components under normal condition, and incomplete amplification pathway components under normal or infection-inflammation conditions. * indicates the C4 cleavage products. (B) The phagocytosis assay of the opsonized beads generated in amplification pathways 1 and 2 under infection-inflammation condition (magnification: 400×). The fluorospheres with PC-BSA (red) or GlcNAc-BSA (green) immobilized on the surface were opsonized by incubating with complete or partial components of the amplification pathway as indicated in the C4 cleavage assay (under infection-inflammation condition). These beads (red or green) were mixed with equal amounts of control beads (green or red) which have BSA immobilized on their surfaces without PC- or GlcNAc-residues. The mixture (opsonized beads∶control beads∶U937 cells) at a ratio of 15∶15∶1 was incubated at 37°C for 30 min with gentle shaking. Cells fixed in 4% paraformaldehyde were visualized under fluorescence microscope. (C) Quantification of the phagocytic efficiency observed under the microscope. Three different fields were chosen for each sample and the average net number of opsonized beads (number of opsonized beads−number of control beads) in each cell in each field was enumerated and calculated. A count of 15 beads in each cell was considered 100% phagocytic efficiency.