[Flow cytometric analysis of immunophagocytosis using sensitized fluorescent microspheres bearing C3b]

We analyzed the phagocytic activity of purified human monocytes using fluorescent latex beads sensitized with IgG or IgG.C3 by flow cytometry. To prepare IgG-sensitized latex beads (BA), BSA-coated latex beads (B) were incubated with diluted rabbit IgG anti-BSA. To bind complement components, BA were incubated with whole serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activity of factor I and C5, resulting in deposition of C1, C4b, C2a, C3b on BA (BAC). Phagocytic activity was assessed by percent phagocytosis and phagocytic index (PI). To eliminate the effects of non-phagocytosed latex beads, subtraction of the data at 4 degrees C from 37 degrees C was performed. Percent phagocytosis for 60 min. was B 5.0%, BA 18.3%, and BAC 57.5%, and PI (ingested latex beads/100 cells) was B 7.9, BA 36.8, and BAC 152.7, respectively. In addition, K-76COOH caused dose dependent inhibition on IgG.C3 mediated phagocytosis. Comparison of inhibition pattern on BAC and BA indicated that K-76COOH directly inhibited C3.C3-receptor binding.