Fitc labeled anti mouse cd8

To determine the effect of therapeutic vaccines on different T cell subsets in mice, blood (100 mL) was collected at the indicated time points in 12 mice/group. Total number of lymphocytes was determined. CD4⁺ and CD8⁺ T cell populations were determined on days 12 and 27 by staining with PE-labeled anti-mouse CD4 and FITC-labeled anti-mouse CD8 antibodies (eBioscience, San Diego, CA). Then CD4⁺ and CD8⁺ T cell populations were measured by flow cytometry. To determine IFN-r⁺ CD8⁺ and CD4⁺ Foxp3⁺ T cell populations, cells were stained with FITC-labeled anti-mouse CD8 or FITC-labeled anti-mouse CD4 (Clone RM4-5, concentration 0.5 mg/m, Biolegend), fixed, permeabilized, and stained with PE-labeled anti-IFN-γ or PE-labeled anti-FoxP3 antibody (Biolegend). T cell subsets were measured using flow cytometry. All experiments were carried out in triplicate.

To evaluate the therapeutic effect of S1P/JS‐K/TMZ/Lipo, the mice were randomly divided into four groups as follows: PBS, JS‐K/Lipo, TMZ/Lipo and S1P/JS‐K/TMZ/Lipo. All the mice were intravenously injected with PBS (100 µL), JS‐K/Lipo (100 µL), TMZ/Lipo (100 µL) and S1P/JS‐K/TMZ/Lipo (100 µL). Tail vein injections treatments were performed every 2 days, for a total of five injections. Body weight was recorded every other day for 16 days. Finally, mice were sacrificed to collect tumors with major organs (heart, liver, spleen, lung, and kidney) to analyze T cell infiltration and tumor apoptosis with different treatments. First of all, the tissue sections of 3 µm thickness from tumors were soaked in acetone for 15 min and then gently washed with PBS. Then, the sections were blocked with 5% BSA and incubated with antibodies against FITC labeled anti‐mouse CD11c (1:250, Biolegend), and FITC labeled anti‐mouse Foxp3 (1:250, Biolegend), FITC labeled anti‐mouse CD4 (1:250, Biolegend), FITC labeled anti‐mouse CD8 (1:250, Biolegend) for 4 h. Next, sections were gently washed three times by PBS and stained with DAPI. Finally, CLSM was applied to collect images.