Impaired Ag uptake in splenic DC from ICSBP^–/– mice. CD11c⁺ s-DCs were purified from ICSBP^–/– and WT mice by magnetic cell sorting. (A) Cells were incubated for 40 minutes with 1 mg/mL DX-FITC or with 0.2 mg/mL OVA-FITC, then washed and analyzed by FACS. Histograms represent the uptake of FITC-conjugated antigens, expressed as percentage of FITC⁺ cells, in samples incubated with the Ag at 37°C and control cells incubated with Ag at 4°C. (B) Splenic DCs were incubated with 1 mg/mL HRP for 10 minutes at 37°C, then washed in cold PBS, cytospun on glass slides, and fixed; the enzymatic reaction was then developed. Slides were counterstained with hematoxylin. Brown spots (indicated by red arrows) reflect the presence of HRP in macropynosomal vesicles. Images were observed through an Achroplan 100 ×/1.25 oil-immersion objective lens. Scale bar represents 50 μm. (C) qRT-PCR analysis of mannose receptor (Mrc1) mRNA in ex vivo s-DCs. Total RNA from ICSBP^–/– and WT s-DCs was used to generate cDNA that was analyzed by qRT-PCR. The cDNA concentrations were normalized to β-actin expression. Fold change of Mrc1 transcripts in WT s-DCs (▪) are expressed relative to those from ICSBP^–/– mice (□) using the ΔΔC_T method (“Materials and methods”). Results are represented as the mean ± SD of 3 separate experiments.