CXCL12 expression in EBV-transformed B cells and its segregation with B-cell phenotype. (A) The expression of CXCL12 and β-actin in LCL cells and hu/SCID tumor cells was evaluated by RT-PCR. Representative results from 5 LCLs and 5 hu/SCID tumors are shown. Lane M corresponds to 50-bp molecular weight marker. Lanes 12 and 13 correspond to a representative case of purified resting B cells and to the same B cells after 48 hours of in vitro stimulation with anti-CD40/IL-4, respectively. (B-C) The intracellular expression of CXCL12 in LCL cells and hu/SCID tumor cells was evaluated by cytofluorimetric analysis. (B) The fluorograms (from top to bottom) show normal B cells not expressing CXCL12, LCL cells, and hu/SCID tumor cells. (C) The expression of CXCL12 in the CD23^low (lower right) and CD23^int (upper right) tumor cell subsets is shown. A representative experiment of 3 consecutive experiments is shown. (D) Confocal microscopic analysis evaluating the coexpression of surface CXCR4 and intracellular CXCL12 in hu/SCID tumors. Cells were fixed, stained with anti-CXCR4 Ab, and subsequently permeabilized before incubation with anti-CXCL12 Ab. The signal for CXCR4 is shown in red, while the CXCL12 signal is in green. Areas of colocalization are shown in yellow.