Figure 5

Flow cytometry analysis of leech nerve cells using anti-human C1qBP antibody (A-D). (A) On the right side, we represent a forward scatter (FSC) versus side scatter (SSC) dot plot for leech nerve cells incubated overnight. Among the cells in the studied gate, 2.86% of autofluorescence cells are shown on the FL1 scale on the left. (B) On the right, we represent an FSC versus SSC dot plot for leech nerve cells incubated overnight alone, then treated with fluorescein-labeled mouse monoclonal anti-human C1qBP antibody for 30 minutes (dilution 1:250 in L-15 medium). In the studied gate, 46.26% of the leech nerve cells are stained as shown on the FL1 scale on the left. The use of the monoclonal antibody did not affect the dot plot profile of the cells. (C) On the right, we represent an FSC versus SSC dot plot for leech nerve cells incubated overnight with 8 μl of rHmC1q supernatant, then treated with fluorescein-labeled mouse monoclonal anti-human C1qBP antibody for 30 minutes (dilution 1:250 in L-15 medium). The fluorescence rapidly decreased and only 5.90% of the nerve cells are stained on the FL1 scale on the left. (D) A flow cytometry histogram overlay shows a shift in fluorescence when the nerve cells are incubated with anti-human C1qBP antibody (red) compared to either nerve cells that were previously incubated with rHmC1q supernatant (blue) or nerve cells alone (gray). These data are representative of three independent experiments. (E) Western blot analysis using polyclonal anti-human C1qBP antibodies from affinity enrichment with biotinylated human C1q. A specific signal corresponding to a 33-kDa molecule, according to the protein ladder (L), was detected when biotinylated human C1q was incubated with leech microglia protein extracts and then eluted on a streptavidin column (1). No immunopositive signal was observed when leech microglial protein extracts were eluted alone on streptavidin column (2) or when biotinylated human C1q was eluted alone with the streptavidin column (3). No signal was detected using the secondary antibody alone as a negative control (data not shown).