Mouse il 10

Mouse tumor tissues were homogenized with extraction buffer. The supernatant was analyzed using mouse IL-10 (Biolegend, 431417) or TGF-β ELISA kits (Biolegend, 433007) according to the manufacturer’s instructions.

To detect the secretion of MCSF or the M1 or M2-phenotype related cytokines, the ELISAs were used with human IL-10 (430604, BioLegend, USA), human TNF-α (430204, BioLegend, USA), TGF-β (DY240-05, R&D, USA), human MCSF (CHE0042-96, 4Abio, China), and mouse MCSF (DY416-05, R&D, USA), mouse TGF-β (88-8350-88, Invitrogen, USA)and mouse IL-10 (431414, BioLegend, USA) kits. Briefly, a 96-well plate was coated with 100 µl diluted capture antibody overnight at 4 °C. Then, 200 µl per well blocking solution was incubated at room temperature for 2 hours. After washing the plate with PBST, a gradient diluted standard, and samples were added (100 µl per well) and incubated at room temperature for 2 h. Then, the plate was washed with PBST, and a diluted detection antibody (100 µl per well) was added and incubated at room temperature for 2 h. After washing the plate with PBST, 100 µl avidin–HRP was added and incubated for 20 min in the dark at room temperature. Then, the plate was washed with PBST, and the substrate TMB (100 µl) was added and incubated at room temperature for 20 min in the dark. A stop solution (50 µl) was then added to terminate the reaction, and the concentration of each sample was detected at 450 nm.