Journal: Blood Advances

Article Title: Absence of complement component 3 does not prevent classical pathway–mediated hemolysis

doi: 10.1182/bloodadvances.2019031591

Figure Lengend Snippet: Comparison of the alternative pathway–mediated hemolysis between the C3-sufficient and C3-Dpl human or C3-deficient rat sera. For the measurements of alternative pathway–mediated hemolysis, Erabb cells were incubated with different concentrations of NHS ranging from 5% to 40%, C3-Dpl human sera (A) or WT C3-deficient (C3KO) rat sera (B) in GVB-Mg++/EGTA buffer, and hemolysis was quantitated by measuring levels of released hemoglobin in the supernatants. (C) In some experiments, Erabb cells were incubated with 100% human or rat sera, and hemolysis was assessed by following the same protocol. These data show almost complete abolition of hemolysis in the C3-Dpl human sera and C3-deficient rat sera. *P < .05.

Article Snippet: In vitro alternative pathway complement–mediated hemolytic assay Rabbit RBCs (E rabb ) (Hemostat Laboratories, Dixon, CA) were incubated with GVB-Mg-EGTA (5 mM barbital, 145 mM NaCl, 0.5 mM MgCl 2 , 10 mM EGTA, and 0.1% gelatin, pH 7.2 ± 0.15; Boston BioProducts) with 5% to 100% of either NHS or C3-Dpl human sera and WT or C3 KO rat sera at 37°C for 20 minutes.

Techniques: Comparison, Incubation

Journal: Journal of Translational Medicine

Article Title: Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway

doi: 10.1186/1479-5876-12-181

Figure Lengend Snippet: Functional hemolytic assays of complement activation. (A) CH50-type antibody-initiated complement assays incubating antibody-sensitized erythrocytes (EA) with 0.2% human serum for 1 hour at 31-41°C showed that hemolysis of antibody-sensitized erythrocytes in human serum differed significantly as a function of temperature (ANOVA P < 0.01). There is 2-fold more hemolysis at 31°C vs. 37°C (* t -test P = 0.01) (P ≤ 0.05 for the comparisons 31 vs. 37, 31 vs. 39, 31 vs. 41, 33 vs. 37, 33 vs. 39, 33 vs. 41, 35 vs. 39, 35 vs. 41, with significantly more hemolysis at lower temperatures) Data are mean ± SEM for 4 independent experiments. (B) Evaluating antibody-initiated complement activation from 0-37°C showed that hemolysis of antibody-sensitized erythrocytes in human serum differed significantly as a function of temperature (ANOVA P < 0.01). There was 2.5-fold more hemolysis at 31°C vs. 37°C (* t -test P = 0.01). (P ≤ 0.05 for the comparisons 31 vs. 37, 24 vs. 37, 19 vs. 37, with significantly more hemolysis at lower temperatures). The trend of increased complement activation was reversed at 13°C. (No statistical difference between hemolysis at 13 vs. 37 or 7 vs. 37). Data are mean ± SEM for 4 independent experiments. (C) AP50-type alternative complement pathway assays, incubating rabbit erythrocytes with 4% human serum in Mg-EGTA-GVBS for 30 minutes at 31-41°C showed that temperature did not influence complement-mediated cell lysis over therapeutic hypothermia temperatures (ANOVA P = 0.45). Data are mean ± SEM for 4 independent experiments. (D) Evaluating alternative pathway activation from 0-37°C showed that alternative pathway activation was significantly inhibited at 24°C and below. There was no difference in the degree of hemolysis at 31°C vs. 37°C) Data are mean ± SEM for 4 independent experiments.

Article Snippet: Alternative complement pathway hemolytic assays were performed by incubating rabbit erythrocytes (CompTech, Tyler, TX) with 4% NHS in Mg-EGTA-GVBS at temperatures ranging from 0°C to 37°C.

Techniques: Functional Assay, Activation Assay, Lysis