Figure 2

Photoreceptor outer segments activate both the classical and alternative complement pathway. (a) Flow cytometry of C3, C4 and C9 neoepitope proteins on the surface of bovine POS following incubation with normal human serum (NHS, shaded area in histograms) vs C3, C4, C9 depleted serum (solid lines in histograms). (b) Diagram of molecular components of the AP and CP/MBL complement pathways. C3 and C5 convertases are indicated in blue and red boxes, respectively. (c,d) Bovine POS were incubated with human sera depleted of various complement proteins and quantified by flow cytometry using a C3-specific antibody (c) or a C9 neo-epitope-specific antibody (d), n = 3. (e and f) Bovine POS were incubated with mouse wt, C3 deficient, C4 deficient, or Ig deficient (Rag2^−/−) serum, surface stained for C3 (e) or C4 (f) protein and quantified by flow cytometry. (g) Flow cytometry analysis of cell-surface complement regulatory proteins and annexin V binding to bovine POS, solid line is isotype control and shaded area is indicated antibody. ^¶p < 0.0001 NHS vs all other sera, ****p < 0.0001 vs C1q/CFD depleted serum, ^#p < 0.001 vs mouse wt serum by 1-way ANOVA with Dunnet’s multiple comparisons test. Error bars are SD for triplicate repeats, the experiments were repeated three times with similar results. NHS = normal human serum, CFD = complement factor D, CFB = complement factor B, P = properdin, MFI = mean fluorescence intensity.