Low attachment 96 well tissue culture plates

Bone marrow-derived macrophage (BMDM) cells were isolated from C57BL/6 or BALB/c mice and induced with 20 ng/mL of GM-CSF (Peprotech, Rocky Hill, CT, USA) for 7 days. Meanwhile, the medium was replaced with fresh medium containing the cytokine and the adherent cells were harvested. Phagocytosis assays were performed by a co-culture of the BMDM macrophages with carboxyfluorescein succinimidyl ester-labeled (CFSE⁺) or GFP⁺ tumor cells at a ratio of 1:4 in a serum-free medium at 37 °C for 4 h, in low-attachment 96-well tissue culture plates (Corning, New York, NY, USA). The cells were harvested and the macrophages were identified by a flow cytometry using anti-F4/80 antibody (eBioscience, San Diego, CA, USA). Then, 7-AAD (eBioscience) was used to exclude the dead cells. The effects of azelnidipine (20 μM) on phagocytosis by BMDM cells were tested, and the anti-CD47 antibody (clone: miap301) were used as the positive control. Phagocytosis rate was determined with the formulation: F4/80⁺GFP⁺ or F4/80⁺CFSE⁺ cells/ Total F4/80⁺ cells.

Murine bone marrow cells were isolated from 7 to 11 weeks old C57BL/6 or BALB/c mice. Human peripheral blood mononuclear cells were collected from venous blood of healthy volunteers, diluted with 2×PBS (pH 7.4) and separated with Ficoll density gradient. The cells were cultured in DMEM (GIBCO, USA) supplemented with 10% fetal bovine serum and 20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF, Peprotech, USA) for 7 days. Meanwhile, medium was replaced with fresh medium containing cytokine, and then the adherent cells were harvested. Phagocytosis assays were performed by co-culture of macrophages with carboxyfluorescein succinimidyl ester (CFSE⁺) or green fluorescent protein (GFP⁺) tumor cells at a 1:4 ratio in serum-free medium at 37°C for 4 hours in low-attachment 96-well tissue culture plates (Corning, USA). The cells were harvested, and primary macrophages were identified by flow cytometry using anti-F4/80 or anti-CD14 antibody (eBioscience, USA). Then, 7-AAD (eBioscience, USA) was added to exclude dead cells in some experiments. Phagocytosis rate was determined as the percentage of CFSE⁺ or GFP⁺ macrophages.