Under deep ether anesthesia, the liver was perfused with Hanks Balanced Salt Solution (HBSS) via the portal vein. Immediately after perfusion, the liver was removed. Tumors were separated from the liver (with a margin of approximately 2 mm from the tumor edge) and minced with scissors. Liver specimens were incubated in 10 ml of HBSS containing 0.05% Type IV collagenase and 0.01 mg/ml DNase I (Sigma). The specimens were shaken for 40 min at 37 °C and then filtered through a stainless-steel mesh (70 µm), suspended in 33% Percoll solution and centrifuged for 15 min at 450 g at room temperature. After the red blood cells were lysed, the remaining cells were washed twice38 (link),39 (link).
For TAM magnetic sorting, single cells prepared as described above were incubated with Fc-blocker and then stained with biotin-conjugated anti-F4/80 Ab (ebioscience) and incubated with streptavidin magnetic beads (Biolegend). Positively labeled cells were collected using a magnet. For monocyte/macrophage magnetic sorting during the monocyte maturation process, biotin-conjugated anti-CD11b Ab (Biolegend) was used, followed by the above-described steps.
For TAM magnetic sorting, single cells prepared as described above were incubated with Fc-blocker and then stained with biotin-conjugated anti-F4/80 Ab (ebioscience) and incubated with streptavidin magnetic beads (Biolegend). Positively labeled cells were collected using a magnet. For monocyte/macrophage magnetic sorting during the monocyte maturation process, biotin-conjugated anti-CD11b Ab (Biolegend) was used, followed by the above-described steps.
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