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Complement activation assay in vitro

YG Yuan Gao
RH Rui Hou
YH Yixin Han
QF Qiaoling Fei
RC Runlan Cai
YQ Yun Qi
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Human whole blood and plasma from healthy volunteers were obtained with written informed consent. The plasma was prepared by centrifuging blood that had been collected in EDTA-coated tubes and was immediately used or frozen at −80 °C until use. Plasma (100 μL, 1:100 dilution) was mixed with 4 μL of CaCl2 (1 M), 4 μL of MgCl2 (1 M), and 10 μL of test substances, and then incubated at 37 °C. After 20 min, the reaction was stopped using 22 μL of EDTA (0.5 M, pH 8.0) and cooling to 0 °C. Tween-80 was used as a positive control28. The level of C5a in the plasma was determined using a commercial ELISA kit according to the manufacturer’s instruction.

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