Fig. 5. Expression of CCR1 protein on the surface of isolated human EVTs. (A-D) Isolated EVTs (7-weeks gestation) were cultured for 4 hours to allow attachment and then double-immunostained with anti-CCR1 mAb followed by rhodamine-conjugated secondary antibody plus either FITC-conjugated anti-cytokeratin 7 mAb (A) or FITC-conjugated anti-vimentin mAb (C). (B) is a higher magnification of the area indicated in (A). (D) is a negative control for (A) in which the anti-CCR1 mAb is replaced by isotype-matched control mAb. About 50% of cytokeratin 7-positve cells (EVTs) are positive for CCR1 (A), which is clearly located on the cell surface (B), whereas CCR1 expression is scarcely detected on the vimentin-positive cells (contaminating villous stromal cells) (C). A control mAb does not stain the surface of cytokeratin 7-positive cells (D). (E) Isolated EVTs (8-weeks gestation) were treated with either anti-CCR1 mAb or isotype-matched control mAb followed by FITC-conjugated secondary antibody and analyzed by FACScalibur. A shift in the fluorescence intensity of the cells treated with anti-CCR1 mAb is observed compared to an isotype-matched control mAb. Scale bars: 50 µm.