Phospho-STAT-5 expression by spleen, PLN, and BM CD8 cells. Untreated B6 mice were killed and, within 10 minutes, single-cell suspensions from spleen, PLN, and BM were fixed in 1.6% formaldehyde phosphate-buffered saline. After permeabilization with 80% methanol and rehydration with 0.5% bovine serum albumin phosphate-buffered saline, cells were stained with anti-TCR PE, anti-CD8 Alexa 488, plus either control mAb or anti-STAT-5(Y694) mAb, both conjugated with Alexa 647. After gating on TCR⁺CD8⁺ cells, the mean fluorescence intensity of phospho-STAT-5 mAb and control mAb were determined on spleen, PLN, and BM samples. (A) Phospho-STAT-5 staining profiles. Representative staining profiles of spleen and BM samples. As a control, spleen cells were incubated for 15 minutes at 37°C with either medium or IL-15 at 25 ng/mL and TCR⁺CD8⁺ cells were analyzed. The scale on x-axis is logarithmic, in arbitrary units, and that on y-axis is linear (maximum value, 55). The numbers represent the mean fluorescence intensity of phospho-STAT-5 mAb (–) and control mAb (–). (B) Phospho-STAT-5 mean fluorescence intensity. Phospho-STAT-5 mean fluorescence intensity values of spleen, PLN, and BM CD8 cells from individual mice and averages are shown in the left panel. In vitro stimulated spleen CD8 cells from individual experiments and averages are shown in the right panel. Average values are shown by the horizontal bar. The panels summarize the results obtained in 7 independent experiments.