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Transwell assays were performed in 24-well plates with inserts (5-μm pore size; Millipore, USA). Briefly, 5 × 104 BMDMs or peritoneal macrophages were cultured in the upper chamber, while the lower chamber contained 600 μl of hypoxic tumor supernatant. After 2 h of incubation at 37 °C, cells that migrated through the polycarbonate membrane were fixed with methanol and stained with crystal violet. The cells were counted in five random fields of view.
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