Fitc anti mouse ifn γ

Every eight weeks, six mice from each group were killed in order to periodically monitor the Tregs and Th cell subsets. The Treg and Th cell subset percentages were measured from the spleen using flowcytometry. Cells that had been harvested from the spleen were stained using antibody markers. Treg detection was done by labelling with PE anti-mouse CD4 (Biolegend), PerCP anti-mouse (Biolegend) and FITC anti-mouse FoxP3 (Biolegend). Th cell subsets, including Th1, Th2 and Th17, were also measured in a similar manner. Prior to staining these cells, cells were stimulated with phorbol myristate acetate (PMA; 50 ng/mL; Sigma, St Louis, MO) and ionomycin (1 μg/mL; Sigma) in the presence of brefeldin A (BD Pharmingen, San Diego, CA) for at least four hours. Then, cells were labelled with PE anti-mouse CD4 (Biolegend). Intracellular staining was performed using PerCP anti-mouse IL-4 (Biolegend), FITC anti-mouse IFN-γ (Biolegend) and PerCP anti-mouse IL-17A to detect Th1, Th2 and Th17, consecutively. All staining was performed according to Biolegend manufacture protocols. All cells were analysed with FACSCalibur (Becton Dickinson).

4- to 6-week-old female BALB/c mice were used for the breast tumor-bearing model. Subcutaneous tumor models were established by inoculating 5 × 10⁵ 4T1 cells into the right flanks of the BALB/c mice. When tumor size reached approximately 100–200 mm³ in volume, the mice were administered physiological saline, PLP (10 mg/kg/d, s.c.) once every 24 h, and TAXOL^® (Harbin Pharmaceutical Group Holding Co., Ltd., Heilongjiang, China, 6 mg/kg, i.p.) and TAXOL^®+PLP once every 4 days. Mice were closely monitored every other day for pain/distress, tumor volume, and body weight. The tumor volume was calculated using the following formula: mm³ = (longest diameter × shortest diameter²)/2. Mice were sacrificed at Day 22. Tumors were removed and weighed, and tumors, spleens, and inguinal lymph nodes were excised for flow cytometer analysis of immune cell subpopulations. Cells were proceeded to be incubated with FITC anti-mouse CD11c, APC anti-mouse CD3, PE anti-mouse CD8a, PerCP/Cyanine5.5 anti-mouse CD4, FITC anti-mouse IFN-γ, PE/Cyanine7 anti-mouse CD11b, and PE anti-mouse F4/80 antibodies (Biolegend, United States) for 30 min at 4°C in the dark. Cellular supernatant was collected for IFN-γ ELISA analysis.