Figure 4. Phenotypic characterization of freshly isolated NK cells from mouse spleen and BM. (A) For all FACS analysis presented herein, cells were gated by doublet discrimination, followed by Live/Dead discrimination, CD45 positivity, and finally, by debris discrimination. Cells were then gated on the CD3−NKp46+ subset for NK cell-subset analysis. FSC (A) and (W), Forward-scatter area and width, respectively; SSC, side-scatter. (B and C) NK cells were gated as CD45⁺NKp46⁺CD3⁻, and percentages and absolute numbers of NK cells were calculated in fresh spleen (B) and BM (C). (D and E) Gates for each surface marker were set by use of unstained or nonspecific mAb isotype controls; a representative staining of each marker is shown. The median fluorescence intensity for each marker was analyzed in spleen (D) and BM (E). Data were analyzed with a 2-tailed, unpaired Student’s t-test (mean ± sd; n = 4–8 mice/group in 3–6 independent experiments; *P < 0.05).