CXCL12 binds to CXCR7 in PB CD34⁺cells. Fresh PB CD34⁺ cells were incubated in serum- and cytokine-free medium supplemented with CXCL12^AF647 (10 ng/mL) for 3 hours and CXCL12-, CXCL11-, CXCR4-, and/or CXCR7- (9C4 and 11G8) blocking antibodies, CXCR7 inhibitors, isotype controls, or CCX704 control. Analysis was performed on a BD Fortessa flow cytometer. (A) Histograms show CXCL12^AF647 binding CD34⁺ cells in each condition. Results are representative of 1 experiment of the 3 performed. (B) Histograms show MFI of cells determined by a BD Fortessa flow cytometer in each experimental condition and expressed as mean ± SD (n = 4). Control was normalized at 100 arbitrary units in each experimental condition. (C) Histograms illustrate CXCL12^AF647 binding inhibition in CD34⁺ cells for each condition. The percentage of CXCL12^AF647 binding inhibition was determined using the formula previously described²⁴ : (1−[MFI−MFI_NC]/[MFI_PC−MFI_NC])*100. MFI corresponds to mean fluorescence intensity of cells incubated with CXCL12^AF647 and CXCL12, CXCL11, CXCR4, and/or CXCR7 (9C4 and 11G8) blocking antibodies or CXCR7 inhibitors; MFI_PC to the cells incubated with CXCL12^AF647 and respective controls; and MFI_NC to cell autofluorescence. Asterisks indicate significance vs control conditions (*P ≤ .05, **P ≤ .01, ***P ≤ .001). Deltas indicate significance between experimental conditions (ΔP ≤ .05, ΔΔP ≤ .01, ΔΔΔP ≤ .001).