Cell surface IL-15 is not associated with its own receptor. (A) MONO-MAC-6 were treated with IFN-γ (500 U/mL) for 24 hours. Cells were then washed and incubated in PBS, acetate buffer (pH 4.4), or trypsin as described in Materials and Methods. All groups were stained with rabbit anti–human IL-15 polyclonal antibodies (Peprotech) and analyzed on FACSCalibur. Fluorescence intensity is represented by open histograms; solid histograms refer to background staining of isotype control. (B) MONO-MAC-6 treated with IFN-γ as described above were incubated with PBS (left panel) or trypsin (right panel). Cells were then incubated with biotinylated human IL-15:Fc fusion protein (Chimerigen; open histogram) or with equal amount of control fusion protein (solid histogram) followed by FITC streptavidin (BD Biosciences). In the central panel, cells were incubated with the biotinylated IL-15:Fc fusion protein and then with acetate buffer, pH 4.4. The x-axis represents the intensity of green fluorescence expressed in a log scale as mean channel, and the y-axis represents the number of cells per channel.