Figure 5.

N-Glycans contributed to hemocyanin binding and incorporation by J774.2 macrophages. A, effect of monosaccharides and CCH, FLH, and KLH on the expression of innate immune receptors analyzed by flow cytometry. J774.2 cells were treated with hemocyanins (50 μg/ml), mannose (Man), or galactose (Gal) (100 mm) for 1 h, and then incubated with an anti-F4/80–Alexa Fluor-647 antibody, as well as primary anti-MR, anti-TLR-4, and anti-MGL following goat anti-rat IgG–FITC serum. Graphs show the MFI of each sample. B, binding/incorporation of hemocyanins to J777-4 cells in the presence of monosaccharides. Representative histograms of J774.2 cells incubated with Alexa Fluor 488–hemocyanins (50 μg/ml) for 1 h in the presence or absence of mannose and galactose (100 mm). C, quantification of the data presented in B. The binding and incorporation of the abovementioned histograms, with the mean fluorescence intensity of cells with hemocyanins and without monosaccharides were defined as 100%. D, binding/incorporation of native and N-deglycosylated hemocyanins. Quantification of the binding and incorporation of hemocyanins, where the mean fluorescence intensity of native hemocyanins was defined as 100%. E, binding of native and N-deglycosylated fluorescent hemocyanins. Representative histograms of D. For all experiments, the viability was assessed using eFluor 780 dye. The data are presented as the mean ± S.E. of four independent experiments. Analysis was by t test. *, p < 0.05; and ****, p < 0.0001.