Splenic Cxcl12 up-regulation in Cxcr4¹⁰¹³-bearing mice. (A) Levels of Cxcl12 mRNAs in total spleen cells from WT, +/1013, and 1013/1013 mice were assessed by real-time quantitative PCR analyses. Each individual sample was run in triplicate with five mice per group, and results (mean ± SEM) are expressed as Cxcl12/Gapdh ratio. (B) Levels (mean ± SEM) of Cxcl12 were determined by ELISA in spleen supernatants from WT, +/1013, and 1013/1013 mice (n = 5 per group). (C–E) Spleen sections from WT, +/1013, and 1013/1013 mice were immunostained for Cxcl12 (C), Tcf21 (D), or both markers (E) in association with DAPI. The color code for antibodies used is shown. Negative controls (iv, C and D) to support the validity of staining were done by adding the secondary antibody alone on 1013/1013 spleen sections. Dashed lines denote the boundary between the white pulp (WP) and red pulp (RP) areas. Arrows indicate the cells that colocalized Tcf21 and Cxcl12. Bars: (C and D) 200 µm; (E) 50 µm. Images are representative of four independent determinations. *, P < 0.05 compared with WT mice (as determined using the two-tailed Student’s t test).