Role of soluble factors and three-dimensional culture in in vitro differentiation of intestinal macrophages

Figure 5 Antigen expression of MO/MAC incubated with IEC in trans-well cultures. Freshly elutriated MO were incubated with the IEC line HT-29 seeded on filter inlays for seven days. Depending on pore size of the inlays only MO or MO and IEC could migrate through the filter. Antigen expression of migrating and non-migrating cells was examined by immunohistochemistry (APAAP-method) and flow cytometry. A: Migrating cells adhered to the plastic surface of the cell culture plate. When filters with a pore size of 3 μm were used, only MO were able to migrate through the membrane. Migrating cells were all negative for the epithelial cell specific marker EP-4 and positive for the MO/MAC-marker CD33; B: Migrating cells showed expression of CD68, CD14, CD16, CD11b and CD11c (left column). Twelve μm pores allowed migration of MO and IEC. None of the tested antigens was expressed by IEC (right column); C: Non migrating cells which remained in the upper compartment of the filter insert were examined by flow cytometry. The CD33-positive cell population (MO/MAC) showed also expression of CD14, CD16 and CD11b; D: Migrating cells which did not adhere to the plastic dish were examined by flow cytometry. Cells showed CD33-expression and were positive for CD14, CD16 and CD11b.