Figure 5.
Figure 5. Endocytosis of FITC-DX and LY by imDCs, macrophages, and mDCs. (A) imDCs were incubated for 30 minutes in the presence (solid lines) or absence (dotted lines) of βig-h3 (10 μg/mL). FITC-DX or LY was then added and incubated for 30 minutes. The cells were washed and analyzed by flow cytometry. Filled histograms represent cells incubated with FITC-DX or LY at 4°C (negative controls). (B) Quantification of imDC endocytosis. The MFIs of 3 independent experiments as shown in panel A are presented as mean ± SD. ▦, endocytosis by unstimulated imDCs; ▪, endocytosis by βig-h3–stimulated imDCs. (C) Inhibition of imDC endocytosis by purified anti–βig-h3 mouse IgG. (Left) imDCs were either untreated (solid line) or preincubated for 1 hour with the affinity-purified βig-h3 antibody (bold line) or nonimmune mouse IgG (dotted line). The cells were then incubated with FITC-DX (20 μg/mL) for 20 minutes and were, on washing, analyzed by flow cytometry. Solid histograms represent cells incubated with FITC-DX on ice. (Right) imDCs were either untreated (○) or pretreated with the affinity-purified anti–βig-h3 antibody (□) or nonimmune mouse IgG (▵) and then incubated with FITC-DX for 10, 20, or 30 minutes before flow cytometry. (D) Macrophages (top panels) and mDCs (bottom panels) were incubated with (solid lines) or without (dotted lines) βig-h3 prior to 30 minutes' incubation with FITC-DX or LY. Filled histograms represent cells incubated with FITC-DX or LY for 30 minutes at 4°C. (E) Quantification of macrophage endocytosis. The MFI of 3 independent experiments as shown in panel D are presented as mean ± SD. ▦, endocytosis by macrophages, which were not prestimulated with βig-h3; ▪, endocytosis by βig-h3–stimulated macrophages. (F) Polymyxin B inhibits LPS-stimulated, but not βig-h3–stimulated, macrophage endocytosis of FITC-DX. (Left) LPS was preincubated with (dotted lines) or without (solid lines) polymyxin B before incubation with macrophages. The macrophages were then incubated with FITC-DX and analyzed by flow cytometry. (Middle) βig-h3 was similarly preincubated with (solid line) or without (dotted line) polymyxin B before incubation with FITC-DX. (Right) CD14 (dotted line) was compared with βig-h3 (solid line) in stimulating macrophage endocytosis of FITC-DX. The filled histograms represent FITC-DX endocytosis by unstimulated macrophages.

Endocytosis of FITC-DX and LY by imDCs, macrophages, and mDCs. (A) imDCs were incubated for 30 minutes in the presence (solid lines) or absence (dotted lines) of βig-h3 (10 μg/mL). FITC-DX or LY was then added and incubated for 30 minutes. The cells were washed and analyzed by flow cytometry. Filled histograms represent cells incubated with FITC-DX or LY at 4°C (negative controls). (B) Quantification of imDC endocytosis. The MFIs of 3 independent experiments as shown in panel A are presented as mean ± SD. ▦, endocytosis by unstimulated imDCs; ▪, endocytosis by βig-h3–stimulated imDCs. (C) Inhibition of imDC endocytosis by purified anti–βig-h3 mouse IgG. (Left) imDCs were either untreated (solid line) or preincubated for 1 hour with the affinity-purified βig-h3 antibody (bold line) or nonimmune mouse IgG (dotted line). The cells were then incubated with FITC-DX (20 μg/mL) for 20 minutes and were, on washing, analyzed by flow cytometry. Solid histograms represent cells incubated with FITC-DX on ice. (Right) imDCs were either untreated (○) or pretreated with the affinity-purified anti–βig-h3 antibody (□) or nonimmune mouse IgG (▵) and then incubated with FITC-DX for 10, 20, or 30 minutes before flow cytometry. (D) Macrophages (top panels) and mDCs (bottom panels) were incubated with (solid lines) or without (dotted lines) βig-h3 prior to 30 minutes' incubation with FITC-DX or LY. Filled histograms represent cells incubated with FITC-DX or LY for 30 minutes at 4°C. (E) Quantification of macrophage endocytosis. The MFI of 3 independent experiments as shown in panel D are presented as mean ± SD. ▦, endocytosis by macrophages, which were not prestimulated with βig-h3; ▪, endocytosis by βig-h3–stimulated macrophages. (F) Polymyxin B inhibits LPS-stimulated, but not βig-h3–stimulated, macrophage endocytosis of FITC-DX. (Left) LPS was preincubated with (dotted lines) or without (solid lines) polymyxin B before incubation with macrophages. The macrophages were then incubated with FITC-DX and analyzed by flow cytometry. (Middle) βig-h3 was similarly preincubated with (solid line) or without (dotted line) polymyxin B before incubation with FITC-DX. (Right) CD14 (dotted line) was compared with βig-h3 (solid line) in stimulating macrophage endocytosis of FITC-DX. The filled histograms represent FITC-DX endocytosis by unstimulated macrophages.

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