Pe anti mouse cd8

The percentages of CD4⁺ and CD8⁺ T lymphocytes subsets and the production of cytokine in the splenocytes of immunized mice were determined using flow cytometry techniques as described previously (Zhang et al., 2015b (link); Wang S. et al., 2017 (link)). Briefly, splenocytes (2 × 10⁶cells/ml) of vaccinated mice were stimulated with G10E peptides (10 μg/mL) for 72 h. Then the suspensions were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE (eBiosciences, USA) for 30 min at 4 °C in the dark.
Splenocytes (2 × 10⁶ cells/ml) were also stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail (eBiosciences, USA) containing Phorbol myristate acetate (PMA, 20 ng/ml), Ionomycin (2 μg/ml), Brefeldin A (1 μg/ml), and Monensin (1 μg/ml) to inhibit the secretion of cytokine into the extracellular space. The cells were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit in accordance with the manufacturer's protocol (eBiosciences, USA) and then stained directly with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC), and anti-mouse IL-10 (PerCP-Cyanine5.5) (eBiosciences, USA) for 30 min at 4°C. All these cell population were analyzed by Cytoflex S Flow Cytometer (Beckman Coulter, USA) and the data were analyzed by CytExpert software.

Madecassic acid (CAS no. 18449-41-7) with a purity of 98.5% was purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd (Taizhou, China). DSS (MW: 36–50 kDa) was purchased from MP Biomedicals Inc. (Irvine, CA, USA). Mouse IL-17 ELISA kit was purchased from Lianke Biotech Co., Ltd (Hangzhou, China). MPO activity assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). FITC-anti-mouse CD3, PE-anti-mouse TCR γ/δ, PE-anti-mouse CD4, PE-anti-mouse CD8, and APC-anti-mouse IL-17A were purchased from eBioscience, Inc. (San Diego, CA, USA). Murine IL-1β and IL-23 were purchased from Sino Biological Inc. (Beijing, China). Mouse TCRγ/δ T Cell Isolation Kit was purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany). TRIzol reagent was purchased from SunShine Biotechnology Co., Ltd (Nanjing, China). PPARγ, phosphorylated (p)-PPARγ, PTEN, p-PTEN, PI3K, p-PI3K, Akt, p-Akt, GSK3β, p-GSK3β, mTOR, p-mTOR, NFATc1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mAb were purchased from HuaBio Inc. (Hangzhou, China). Cyclosporin A, GW9662, MK-2206, and rapamycin were obtained from CSNpharm (Chicago, USA). Rosiglitazone, FK506, LY294002, and SC-97 were purchased from Selleck (Houston, USA).