Analyses ofthe classical pathway–mediated hemolysis in a syngeneic experimental system using preassembled C4b2a. (A) For measuring classical pathway–mediated hemolysis in a syngeneic experimental system, antibody-sensitized rat RBCs were prepared using an anti-rat antiserum, then incubated with different concentrations of WT or C3-deficient (C3KO) rat sera in GVB⁺⁺ buffer, and hemolysis was quantitated by measuring levels of released hemoglobin in the supernatants. These data show significantly reduced classical pathway–mediated hemolysis in the absence of C3 when lower concentrations of sera were used but comparable and almost complete hemolysis when higher concentrations of sera (50%-100%) were incubated. (B) In other experiments, the classical pathway C3 convertases were assembled on E^shA cells after incubation with C3-Dpl sera in the presence of the potent C5 inhibitor SSL7 to suppress hemolysis. Then the E^shA cells with preassembled C4b2a were incubated with sera from different WT and C3 KO rats (n = 6 in each group). Hemolysis was measured to assess the activities of C4b2a in directly activating C5. These results show that the preassembled C4b2a caused almost completely hemolysis in the presence and absence of C3, demonstrating that C4b2a can directly activate C5 in the absence of C3. *P < .05.