Endometrial cancer tissues were digested in ImmunoCult‐SF Macrophage Medium (STEMCELL Technologies) containing 1 mg/ml collagenase IV (Sigma‐Aldrich) for 10 min at 37°C at a speed of 200 rpm. Individual cells from EC tissues were centrifuged after filtering using a sterile nylon mesh (70 μm; Falcon). Cell particles were resuspended in 100 μl PBS and sorted using anti‐CD45, CD1c, CD15, CD20, CD11b, and CD206 (Miltenyi Biotec) Abs for 40 min at 4°C. After washing with PBS, the cells were resuspended in PBS for subsequent sorting (FACSAria II; BD Biosciences). The primary TAMs were sorted using CD45+CD1c−CD15−CD20−CD11b+CD206+ cells.
The dissected clinical EC tissues were digested in RPMI‐1640 medium (Gibco) containing 1 mg/ml collagenase IV type (Sigma‐Aldrich) and incubated at 37°C for 10 min (200 rpm). The digested single cells of the EC tissues were filtered and pelleted. Cells were resuspended in 100 μl PBS, and the cells were incubated with anti‐human CD45 (Miltenyi Biotec) and CD3 (BioLegend) Abs at 4°C for 40 min. After washing, the cells were resuspended in PBS and sorted (FACSAria II; BD Biosciences) using the CD45+CD3+ sorting strategy.
The dissected clinical EC tissues were digested in RPMI‐1640 medium (Gibco) containing 1 mg/ml collagenase IV type (Sigma‐Aldrich) and incubated at 37°C for 10 min (200 rpm). The digested single cells of the EC tissues were filtered and pelleted. Cells were resuspended in 100 μl PBS, and the cells were incubated with anti‐human CD45 (Miltenyi Biotec) and CD3 (BioLegend) Abs at 4°C for 40 min. After washing, the cells were resuspended in PBS and sorted (FACSAria II; BD Biosciences) using the CD45+CD3+ sorting strategy.