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. 2011 Apr 6;6(4):e18275. doi: 10.1371/journal.pone.0018275

Figure 5. Flow Cytometry of rCR2 or anti-C3d bound to C3d+ red blood cells.

Figure 5

Sensitised sheep red blood cells were incubated with human C6 deficient serum to generate C3d deposition on RBC in the absence of cell lysis. (A) To confirm C3d deposition, C3d+ RBC incubated with anti-human C3d (black line) or isotype control (solid grey) followed by mouse anti-human-FITC secondary. (B) To demonstrate rCR2 binding to C3d, C3d+ RBC cells were incubated with rCR2 (black line) or an irrelevant His-tag protein, C2A, (solid grey) followed by secondary anti-HIS-FITC. (C) To further demonstrate that the mutant K41E CR2 mutant did not bind to C3d, C3d+ RBC cells were incubated with rCR2 (black line) or K41E CR2 (solid grey) followed by secondary anti-HIS-FITC. (D) To demonstrate that rCR2 specifically bound to C3d, C3d+ RBC cells were first incubated with the inhibitory anti-human C3d prior to incubation with rCR2 and secondary anti-HIS-FITC (solid grey) or as positive control rCR2 and secondary anti-HIS-FITC alone (black line).