100 μL of suspension fluid was added to 0.25 μg APC anti-mouse F4/80 antibody (BioLegend), 1.0 μg PE anti-mouse CD86 antibody (BioLegend), and 0.125 μg FITC anti-mouse CD206 antibody (BioLegend), respectively. Homogenized solutions were placed in the dark at 4°C for 30 minutes, followed by addition of 200 μL FACS buffer, and centrifuged at 4°C, 300 ×g for 5 minutes. After removal of supernatant, the cellular solutions were washed with FACS buffer, followed by addition of 1 mL FACS buffer to resuspend cellular samples. Finally, cellular aggregates were broken up and analyzed with BD FACSCanto II, APC anti-mouse F4/80 antibody-specific M1 macrophage (F4/80+), APC anti-mouse F4/80 antibody, and FITC anti-mouse CD206 antibody-specific M2 macrophage (F4/80+, CD206+). FlowJo software was used to analyze the percentage of macrophages in the lung tissues and the proportions of M1 and M2 macrophages in the lung and tumor tissue samples.
Percentage of macrophages: macrophages (F4/80+)/cells.
Percentage of M1 macrophages: M1 (F4/80+, CD86+)/macrophages (F4/80+).
Percentage of M2 macrophages: M2 (F4/80+, CD206+)/macrophages (F4/80+).
Wang W.J., Wu Y.S., Chen S., Liu C.F, & Chen S.N. (2015). Mushroom β-Glucan May Immunomodulate the Tumor-Associated Macrophages in the Lewis Lung Carcinoma. BioMed Research International, 2015, 604385.
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