Circulating CLL cells that recently emigrated from the LN in vivo show upregulation of classical/alternative NF-κB signaling. NF-κB nuclear localization was examined in FFWB samples of patients with CLL using imaging FCM (ImageStream). (A) Representative cell images displaying staining for CD5, CD19, CD69, CXCR4, RelA/RelB/p100,52, 7AAD, 7AAD/RelA, 7AAD/RelB, or 7AAD/p100,52 (merge) in a CLL sample. The images were captured at ×60 magnification. (B) Fold difference in nuclear localization of RelA, RelB, and p100,52 in CD69⁺/CXCR4^Low CLL (CD5⁺/CD19⁺) cells as compared with CD69⁻/CXCR4^High CLL cells in multiple patient samples. Statistical significance was determined by Student t test. *P < .05; **P < .01; ***P < .001. Data are presented as mean ± SD.