Ebioscience kit

Tumor and spleen tissues were collected for lymphocyte isolation. The colon was cut longitudinally, washed with PBS, and 1–2 cm of colon tissue surrounding the tumor was excised. After removing epithelial cells and the mucus with solution A [50 mL of PBS, 3 mL of 0.5 mM EDTA, 500 μL of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 25 μL of 2 M dithiothreitol (DTT)], the colon tissue was transferred to a new centrifuge tube. After addition of solution B (50 mL of PBS, 3 mL of 0.5 mM EDTA, and 500 μL of 1 M HEPES), the colon tissue was digested with 1 mg/mL collagenase VIII (Sigma–Aldrich), followed by filtration and resuspension of the cell precipitate in a 40% Percoll solution. Next, the cell precipitate was washed with 80% Percoll and resuspended in PBS to obtain a single-cell suspension. The cells were incubated with antibodies (Supplementary Table S4) and stained with propidium iodide. Flow cytometry was performed using an LSRFortessa X-20 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Single-cell suspensions from spleen tissue were prepared and analyzed using the same method. CD4⁺CD25⁺FOXP3⁺ and other cell populations were characterized using an eBioscience kit (eBioscience, San Diego, CA, USA).