Texas red conjugated zymosana

In vitro. Texas Red-conjugated zymosan A (S. cerevisiae) bioparticles (Z2843, Life Technologies) were used for phagocytosis assay as previously described [54 (link)]. Briefly, zymosan bioparticles were incubated with purified zymosan-specific rabbit polyclonal IgG antibodies for 1 h at 37°C for complex formation. Meanwhile, sorted CD11b⁺F4/80⁺ IL-13Rα1⁺ and IL-13Rα1⁻ macrophages (10 × 10⁴/well) were cultured in 96-well plates for 1 h at 37°C for adherence. The opsonin-bound bioparticles (1 × 10⁴/well ) were then transferred to the culture and the incubation continued for 3 hrs. The cells were then detached and surface attached bioparticles were quenched by trypan blue solution. Phagocytized bioparticles were analyzed by flow cytometry.
In vivo. Texas-Red zymosan bioparticles (1 × 10⁴ bioparticles in PBS/mouse) were injected intravenously into IL-13Rα1^+/+-GFP mice for complement receptor-mediated internalization. After 24 hrs, splenocytes were harvested, stained with anti-CD11b, anti-CD11c, and anti-F4/80 antibodies and CD11b⁺ F4/80⁺IL-13Rα1⁺ macrophages were analyzed for presence of Texas-Red bioparticles by flow cytometry in comparison with CD11b⁺ F4/80⁺IL-13Rα1⁻ macrophages.