Proliferation and survival of miR-155 tg vs wt NK cells. (A) BrdU drinking water was administrated daily for 10 days to wt and miR-155 tg mice. BrdU incorporation in splenic NK cells was determined by surface staining of NK1.1 and CD3, followed by intracellular staining of BrdU. The histogram represents BrdU incorporation within a gated population of NK1.1⁺CD3⁻ wt and miR-155 tg NK cells. Mean ±SEM of percentage of BrdU incorporation of wt and miR-155 NK cells from 10 mice is shown on graph (right panel). (B) Left: 2 × 10⁴ FACS-sorted NK1.1⁺CD3⁻ splenic NK cells were cultured in IL-15 (100 ng/mL) for 7 days. Viable cells were enumerated after culturing for 3, 5, and 7 days by trypan blue dye exclusion. Right: eFluor 670–labeled NK1.1⁺CD3⁻ wt and miR-155 tg NK cells were cultured with IL-15 for 11 days. Viable cells were assessed for proliferation by measuring serial dilution of dye fluorescence. (C) Splenocytes of wt, miR-155 tg, IL-15 tg, or double miR-155/IL-15 tg mice were stained with anti-NK1.1 and anti-CD3 Abs and analyzed by flow cytometry for percentage of NK1.1⁺CD3⁻ NK cells (left) and for absolute number of NK cells (right). (D) Freshly isolated splenocytes were cultured in medium without cytokines for 24 hours, followed by surface staining of NK1.1 and CD3 and labeling with 7-AAD and annexin V. Representative dot plots from 5 experiments show staining for 7-AAD and annexin V in NK1.1⁺CD3⁻ NK cells. *Statistically significant; see text for details.