Figure 4

Crosstalk between 4T1 cells and M2-polarized macrophages stimulates cytokine production. (A) 4T1 cells and/or M2-polarized macrophages (M2-Mϕs) were plated in 12-well plates, and 24-hour-conditioned media (CM) were collected. Four independent experiments were conducted and the CM were pooled. The relative levels of cytokines in the pooled CM were estimated using a mouse cytokine array panel A kit. Expression levels were normalized to the levels of the positive control spots (contained within the membrane), and the positive control levels were set at 100%. (B) 4T1 cells or M2-Mϕs were plated on cell culture coverslips (24 mm × 30 mm) in eight-well plates. After 24 hours, coverslips (containing 4T1 cells or M2-Mϕs) were placed on the same wells of four-well plates (4T1 + 4T1; 4T1 + M2-Mϕs; or M2-Mϕs + M2-Mϕs) for 24 hours. Total RNA was isolated, and real-time RT-PCR was performed. Results are shown as means ± standard error of the mean of three independent experiments performed in duplicate. Each of the control levels (4T1 cells alone or M2-Mϕs alone) was set to 1. G-CSF, granulocyte colony-stimulating factor; IP-10, IFNγ-induced protein-10; KC, keratinocyte chemoattractant; M-CSF, macrophage colony-stimulating factor; MCP-1, monocyte chemotactic protein-1; MIP-1α, macrophage inflammatory protein-1 alpha; RANTES, regulated upon activation, normal T-cell expressed and secreted.