NK cell potential of Thy-1⁺ cells present in mixed colonies. Sca-1⁺c-kit⁺ cells were purified from C57BL/6-Ly5.1/C57BL/6-Ly5.2 F1 (Ly5.1/Ly5.2) mice. Cells were cultured in the methylcellulose media containing IL-2, IL-7, IL-11, and SF. After incubation for 18 days, mixed NK cell colonies were individually picked, pooled, stained with PE-anti–B220 and FITC-anti–Thy-1, and analyzed by flow cytometry (A). Thy-1⁺B220⁻ cells (R1) were sorted and incubated in hanging drop for 20 hours with fetal thymus lobes from BDF1 (Ly5.2) mice. The lobes were transferred to filter membranes and cultured for 11 days. Cells recovered from lobes were stained with FITC-anti–Ly5.1 and PE-anti–NK1.1, PE-anti–CD3 or mixture of PE-hamster–IgG and PE-mouse IgG_2a and analyzed by FACS Vantage (B).