CpG can be loaded into tumor cells independently of TLR9 and leaked into the immediate microenvironment. (A) H11, EL4, or TLR9KO tumor cells were incubated with CpG-FITC. Cells were thoroughly washed and analyzed by flow cytometry. Results were consistent across 3 independent experiments. (B) Representative confocal microscopy image of tumor cells loaded with CpG-FITC for 24 hours. Blue = DAPI, red = CD19, and green = CpG-FITC. (C) CpG/H11 tumor cells or media were plated in the upper portion of a permeable supports. Either C57BL/6 or TLR9KO macrophages were plated as responder cells in the base of the permeable supports. After 24 hours, macrophages were collected and CD40 and CD80 expression were determined by flow cytometry. All plots were gated on live cells.