Fig 4: Assessment of AP complement activity in Masp3^−/− mice using zymosan-based and red blood cell-based C3b opsonization assays.

(a) Representative FACS analysis of C3b opsonization of zymosan particles (gated for >98% events on forward and side scatter plots) after in vitro incubation with 20% plasma from wildtype mice (WT, showing 3 out of 7 mice tested), Masp3^−/− mice (Masp3KO, showing 3 out of 8 mice tested) or FD knockout mice (fDKO, showing 2 out of 4 mice tested). C3b opsonization by Masp3^−/− mouse plasma was markedly reduced compared with that of wildtype mouse plasma but did not reach background levels of FD knockout mice. (b) Quantitation of % zymosan particles with C3b deposition reaching to a specified range as defined by the horizontal lines in panel (a). Each symbol represents data from an individual mouse plasma sample. WT (n=7), Masp3KO (n=8) and fDKO (n=4). (c) Survival of CFSC-labeled CD55^−/−/Crry^−/−/C3^−/− mouse erythrocytes when transfused into wildtype (WT), Masp3^−/− (Masp3KO) or FD knockout (fDKO) recipient mice (n=4 recipients for each group). Elimination of CD55^−/−/Crry^−/−/C3^−/− mouse erythrocytes in wildtype recipients was mediated by EVH after AP complement-dependent C3b opsonization of the cells (31). EVH of CD55^−/−/Crry^−/−/C3^−/− mouse erythrocytes was largely prevented in FD knockout mouse recipients but only moderately ameliorated in Masp3^−/− mouse recipients. Percentage of transfused cells remaining at various time points was normalized to that at 5 min after transfusion (31).