Flow cytometric analysis of the expression of chemokine receptors in transfected HEK 293 cells. Twenty micrograms of FLAG epitope-tagged CHEMR1, CCR-4, and CCR-1 and 0.5 μg of CMV-luciferase gene were transfected into HEK 293 cells. After 12 hours, 2 × 105 cells were washed, resuspended in HBSS, and mixed with 1 μg of either anti-FLAG MoAb (M2) or mouse IgG1 isotype control. After washing the cells, the cells were incubated with goat antimouse IgG-FITC, washed, and subjected to flow cytometric analysis. The shaded areas indicate the isotype control, whereas the lines denote fluorescence reflecting expression of the chemokine receptors. Each 2 × 105 transfected cells produced approximately 10,000 light units of luciferase activity; therefore, the fluorescence intensity shown above represents a quantitative difference of the surface expression of each receptor due to compensating the difference of transfection efficiency. At this condition, the mean fluorescence intensities (MFI) for CHEMR1, CCR-4, and CCR-1 were 127.7, 24.9, and 91.2, respectively.
