CXCR7 is mainly expressed in intracellular compartments and is partly co-localized with CXCR4 in PB CD34⁺cells. (A) The CXCR7 protein expression level in Lin^– immunomagnetically selected cells was analyzed on a FACScalibur flow cytometer after labeling with specific antibodies. Results are representative of 1 experiment of the 3 we performed. (B-C) PB CD34⁺ cells were purified immediately after density gradient separation (Inc^–) or after incubation on a plastic support (Inc⁺). The CXCR4 and CXCR7 extracellular and intracellular expression were analyzed on a FACScalibur flow cytometer after labeling with specific antibodies. Results are representative of 1 experiment of the 3 to 6 performed. Histograms show the percentage of CXCR4- and CXCR7-expressing CD34⁺ cells and are expressed as mean ± SD. ***Significance between experimental conditions (P ≤ .001). (D) The CXCR4 and CXCR7 mRNA expression levels in freshly purified PB CD34⁺ cells (Inc^–) were analyzed by quantitative RT polymerase chain reaction. The expression levels of each receptor are normalized on the TBP housekeeping gene by a relative quantification. ns, no significance between both receptors. Results are representative of 1 experiment of the 7 performed. (E) PB CD34⁺ cells were incubated for 45 minutes in a serum- and cytokine-free medium in the presence or absence of CXCL12 (0.5 ng/mL). The CXCR4 and CXCR7 protein expression of cells were analyzed by a TC5 SP5 confocal microscope (Leica, Wetzlar, Germany) after being labeled with specific antibodies. The scale bar represents 10 µm. (F) KG1 cells (4.10⁷ ) were incubated with or without CXCL12 (0.5 ng/mL) for 5 minutes, lysed, immunoprecipated with anti-CXCR4 or anti-CXCR7 antibodies, and immunoblotted with anti-CXCR7 antibody.