Pe anti mouse foxp3

Mice were sacrificed and tumors were harvested. 0.3 mg tumor tissue was isolated and added into a solution of collagenase (400 units per mL), DNase 1 (100 μg mL⁻¹), and hyaluronidase (0.04 units per mL). The mixture was shaked under 37 °C for 1 h. After, the mixture was passed through a 70 μm nylon cell strainer. The sample was washed 3 times with PBS (with 2% FBS) and then resuspended in 1 × 10⁶ cells per mL for flow cytometry analysis. Antibody (AF488-anti mouse CD45, PE/Cy7 anti-mouse CD3, APC anti-mouse CD4, BV510-anti mouse CD8, PE anti-mouse FOXP3 and PE anti-mouse IFN-γ, all purchased from BioLegend) was added following the antibody manufacturer's recommendations and incubated for 30 min on ice. Cells were analyzed on BD FACS Celesta, and data were analyzed using FlowJo 10. Cellular events were first gated by forward and then by side scatter characteristics. The cell counts for the CD45⁺, CD45⁺CD3⁺, CD45⁺CD3⁺CD4⁺, CD45⁺CD3⁺CD4⁺FOXP3⁺ CD45⁺CD3⁺CD8⁺ and CD45⁺CD3⁺CD8⁺IFN-γ⁺ T lymphocytes were assessed. For quantification, the proportion of each immune subpopulation was determined by flow cytometry. This number was then multiplied by the total number of cells in the tumor, which had been determined before flow cytometry by counting with a hemocytometer. To determine cell density, cell counts were normalized to tumor weight (cell number per gram tumor).