Fig. 11

Activation of DC and T lymphocytes by Nano-DOX-treated 4T1 cells. a–d Immunostaining of CD40, CD80, CD83 and MHC-II (markers of DC activation) in bone marrow-derived DC (BMDC) assayed by FACS. Representative FACS histograms are presented in Additional file 1: Fig. S6. e Spleen-derived CD4⁺ and CD8⁺ T lymphocytes characterized by immunofluorescent staining and FACS.f, g Proliferation of CD4⁺ and CD8⁺ T lymphocytes, assayed by CFSE staining and FACS. Representative FACS histograms are presented in Additional file 1: Fig. S7. h, i Immunostaining of CD69 (a marker of T lymphocyte activation) in CD4⁺ and CD8⁺ T lymphocytes, assayed by FACS. Representative FACS dot plots are presented in Additional file 1: Fig. S8. j Immunohistochemical staining of CD4, CD8, CD69 and foxp3 (a marker of Treg) in 4T1 tumor xenografts at the end of 3-week treatment of Nano-DOX or DOX. In the ex vivo cell experiments, BMDC were first treated with Nano-DOX (2 μg/mL) or culture medium conditioned by Nano-DOX (2 μg/mL)-treated 4T1 cells (ND-CM) for 24 h. Spleen-derived lymphocytes were then co-cultured with the BMDC in the same system for another 24 h. In FACS analysis, geometric means were used to quantify fluorescence intensity. Values were mean ± SD (n = 3, *p < 0.05, **p < 0.01)