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. 2017 Sep 25;8:1188. doi: 10.3389/fimmu.2017.01188

Figure 2.

Figure 2

Binding of FXII to apoptotic cells is dependent on phosphatidylserine (PS). (A) After preincubation with or without annexin V at the indicated concentrations in the presence of 2.5 mM CaCl2 for 20 min, apoptotic CEM cells were labeled with 100 nM B-FXII and PE-avidin as described above. The binding of FXII was analyzed by flow cytometry and indicated as a percentage compared to the binding in the absence of annexin V and CaCl2, which was set to 100% (n = 4). ***p < 0.001. (B) B-FXII (100 nM) was preincubated with or without 1 mM phosphatidylcholine (PC) or PS liposomes at 4°C for 15 min, and then apoptotic cells were added. After incubation for 60 min, the binding of B-FXII to apoptotic cells was analyzed by flow cytometry as described above, and shown as the mean fluorescence intensity (MFI). **p < 0.01. (C) FXII binds to phosphatidylserine in a surface plasmon resonance assay. Serial concentrations of FXII were flowed over PS or PC liposomes immobilized on the Biacore sensor chip. The response curve of PS/FXII binding was obtained by subtracting the curve of PC from that of PS. Curves were analyzed with BIAevaluation software. The KD was 3.857E-9 M, and the Rmax was 787.0 Ru.