Tumor Cell Isolation from Macrophages

E0771-GFP tumor cells were plated either alone or in direct contact with BAC1.2F5 macrophages at a 1:4 ratio (200,000 tumor cells and 800,000 macrophages) for 48 h in DMEM supplemented with 10% FBS. The following day, tumor cells and macrophages were collected and tumor cells were isolated from macrophages using MACS CD11b Microbeads (Miltenyi Biotec, cat #130-049-601), as previously described^{101 (link)}. Briefly, after counting, 1 × 10⁷ cells were resuspended in 90 µL of buffer (PBS, 0.5% BSA, and 2 mM EDTA) and 10 µL of MACS CD11b Microbeads (Miltenyi Biotec). These microbeads are colloidal super-paramagnetic beads conjugated with monoclonal anti-mouse CD11b (Mac-1α) antibodies. The cells were placed at 4 °C for 15 min and then placed in a magnetic separator to separate macrophages from tumor cells. The purification of tumor cells from macrophages is in a range of 95%, as we previously described^{101 (link)}. Collected tumor cells were counted immediately and intravenously injected in mice bearing a lung window.