Isolated Tax tumor cells express transcripts for osteoclast-activating factors. (A) Single-cell suspensions of 3 Tax⁺ tumors were sorted by FACS according to FcγR II/III (CD16/CD32) FITC-negative, -low, and -high intensity. Expression of the Tax gene was detected in CD16/CD32-high but not in -negative cell populations by RT-PCR. GAPDH was used as an internal control. (B) Cytospin and Wright-Giemsa-Grünwald staining was performed on sorted cells. CD16/CD32 FITC-negative cells were normal lymphocytes/stromal cells. CD16/CD32 FITC-high cells were tumor cells, whereas CD16/CD32 FITC-low cells were neutrophils (× 20). (C) CD16/CD32 FITC-high cells were immunofluorescently stained with Tax antibody (green, × 20). (D) RT-PCR of selected osteoclast-activating factors in CD16/CD32 FITC-negative (N) and FITC-high (H) cells was performed on the mRNA of purified cell types isolated from whole tumors. RANKL was expressed in CD16/CD32 lymphocytes/stromal cells; IL-6, IL-1α, and M-CSF were expressed in Tax tumor cells; IL-1β, TGFβ, PTHrP, and TNFα were expressed in both CD16/CD32-negative and -high cell populations.