In vitro development of NK cells from the spleen-cell culture supplemented with rmIL-2.

Spleen cells derived from +/+, mi/mi, and tg/tgmice were cultured in α-MEM continuously supplemented with rmIL-2. On days 7 and 10, nonadherent cells of the culture were harvested and examined by immunologic (panel A), morphological (panel B) and molecular biologic (panel C) methods. Three independent experiments yielded similar results. (A) FACScan analysis to detect NK1.1 marker expression in the cultured spleen cells on day 10. Gates indicate the population of NK1.1⁺ cells in the culture. (B) Morphological detection of NK cells. Cytospin preparation of the culture was done on days 7 and 10, and cells were stained with Giemsa solution. Through days 7 to 10, azurophilic cytoplasmic granules were well developed in large lymphocytes of both +/+ andtg/tg mice, but not of mi/mi mice. (C) Northern blot analysis to detect the Gr B, perforin, and MITF gene expression in the spleen-cell culture. Total RNA (5.0 μg) was extracted from the aliquots of the cultured cells on day 10, blotted, and hybridized with the Gr B, perforin, or MITF probe. Reprobing with β-actin verified an RNA equal loading.