2.10. Detection of immune cells by flow cytometry

In this study, the animals in group 1 and group 3 were sacrificed on day 21. Tumor tissue specimens were extracted from nude mice, cut into pieces, and immediately mixed with PBS (0.01 M, pH 7.4). The mixture was filtered using a 300 μm nylon net to harvest tumor cells. Then, the suspension was washed twice with PBS (0.01 M, pH 7.4) after centrifugation (800–1000 r/minutes, five minutes). The precipitate was then resuspended in 1 mL of NS. Fluorescent antibodies were added to the suspension at 4 °C for 15 minutes and then analyzed by FCM. Flow cytometry was used to detect CD206, iNOS, F4/80, CD8, CD28, CD4, and Foxp3. These markers are mainly targeted at M2, CTL, and Treg immune cells.