After treatment, immune cells were isolated from tumor tissues or tumor draining lymph nodes of each group of mice (n = 6) for immune response analysis. Typically, tumor draining lymph node DCs were isolated using RPMI 1640, cell filters (70 μm, BD FALCON), and stained with anti-CD45, anti-CD83, anti-CD80, and anti-MHCⅡ. Finally, cells were washed once with cell staining buffer and analyzed by flow cytometry (BD FACS Calibur, United States).
Tumor-associated macrophages (TAM) and cytotoxic T lymphocytes (CTL) were isolated from tumor tissues using RPMI 1640, collagenases I and III (1 mg/ml), red blood cell lysis buffer, and cell filters (70 μm, BD FALCON). Then, each sample was divided into two samples for TAM and CTL analysis respectively. For TAM analysis, samples were stained with anti-CD45, anti-F4/80, anti-CD86, and anti-CD206. For CTL analysis, samples were stained with anti-CD3, anti-CD8, and anti-CD4. Then, cells were flushed with cell staining buffer and analyzed by flow cytometry (BD FACS Calibur, United States).
Tumor-associated macrophages (TAM) and cytotoxic T lymphocytes (CTL) were isolated from tumor tissues using RPMI 1640, collagenases I and III (1 mg/ml), red blood cell lysis buffer, and cell filters (70 μm, BD FALCON). Then, each sample was divided into two samples for TAM and CTL analysis respectively. For TAM analysis, samples were stained with anti-CD45, anti-F4/80, anti-CD86, and anti-CD206. For CTL analysis, samples were stained with anti-CD3, anti-CD8, and anti-CD4. Then, cells were flushed with cell staining buffer and analyzed by flow cytometry (BD FACS Calibur, United States).