Intracellular expression and secretion of CXCL12 and its role in monocyte to macrophage differentiation. (A) Intracellular expression of CXCL12 in monocytes (primary or THP-1 cell line) or lymphocytes (resting or HSB-2 T cell line) detected by flow cytometry in permeabilized cells. Control isotype (filled histogram) and CXCL12 (empty histogram) are shown in a representative experiment of 3. (B) ELISA quantification of the secreted CXCL12 during 48 hours of culture by peripheral blood monocytes, monocytic cell lines (THP-1 and U-937), and lymphocytes (resting and HSB-2). Mean ± SD; n = 3. (C) CD163 membrane expression on peripheral blood monocytes treated for 48 hours with CXCL12 (100 ng/mL), CXCR4 antagonist (AMD3100; 25 μg/mL), CXCR7 antagonist (CCX733; 100nM) or untreated ⁽⁻⁾ was analyzed by flow cytometry. Mean fluorescence intensity (MFI) of positive cells was determined and fold change regulation relative to untreated control cells is represented. Mean ± SD; n = 4. (D) M-CSF ELISA quantification of culture supernatants of peripheral blood monocytes treated with AMD3100 and/or CCX733 at the concentrations previously indicated during 48 hours. Mean ± standard error; n = 3; *P < .05. (E) CD14, CD4, and CD163 expression analysis by flow cytometry of M-CSF–derived macrophages (5 days) in the absence (control, shaded profile) or in the presence of CXCL12 (100 ng/mL, black line). A representative experiment of 3 is shown.