Fitc anti il 17a

Fluoxetine hydrochloride (Tocris Bioscience, USA) was reconstituted in phosphate buffered saline (PBS). Digoxin (Sigma, USA) was dissolved in dimethyl sulfoxide and further diluted appropriately in PBS to get the desired concentration at the time of injection. Peptides viz. MOG(35-55), PLP(131-151) acquired from Anaspec, USA used for disease induction were dissolved in PBS and emulsified in Freund's complete adjuvant (Sigma, USA). Antibodies anti-CD3, anti-CD4, PE-anti-TCRβ, APC-anti-IFNγ, FITC-anti-IL-17A, anti-IL-4, anti-IL-23, Alexa Fluor 488-anti-rat, Alexa Fluor 555-anti-rabbit, cytokines, IL-12, IL-17A, IL-21, IL-23, TGFβ, IL-6 were purchased from BD Pharmingen, Abcam, Cell Signaling Technology, and RnD Technologies. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (certified) were procured from Gibco, USA.

After Fc receptors blocking (BD Biosciences, USA), an appropriate concentration of the fluorescence-labeled antibody was used for the staining of surface antigens at 4 °C for 30 min in dark place. Fluorochrome-conjugated monoclonal antibodies of cellular markers: PercpCy5.5-anti-CD3, FITC-anti-IL-17A, PE-anti-CD4, PE-Cy7-anti-NK1.1 (BD Bioscience, USA). FITC-anti-CD69, FITC-anti-IFN-γ; PE-anti-FasL, PE-anti-TRAIL, PE-anti-CD107a, and APC-anti-NKG2D (eBioscience, San Diego, CA). The intracellular cytokine staining, including INF-γ and IL-17A, was using Mouse Intracellular Cytokine Staining Starter Kit (BD Biosciences, USA), according to the kit’s instructions. Samples were measured by a BD Accuri C6 plus flow cytometer (BD Biosciences, USA), and the data were managed using BD Accuri C6 plus analysis (BD Biosciences, USA).

The standard of berberine (C₂₀H₁₈NO₄, molecular weight: 235.324 kD) was obtained from Solarbio Biotechnology Co., Ltd. (Beijing, China). Dextran sulfate sodium (DSS molecular weight: 36000-50000kD), ciprofloxacin, metronidazole, Phorbol 12-myristate (PMA), ionomycin, and Bravertin A were also obtained from Solarbio Biotechnology Co., Ltd. (Beijing, China). Total DNA extraction kit, total RNA extraction kit, first-stand cDNA reverse transcription kit, polymerase chain reaction kit, and primers were obtained from TianGen Biotechnology Co., Ltd. (Beijing, China). Mouse IL-10 and IL-17 ELISA kits were obtained from Multi Science Biotechnology Co., Ltd. (Hangzhou, China). APC anti-CD4, FITC anti-IL17A, PE anti-CD25, and Alexa Fluor 488 anti-Foxp3 antibodies for flow cytometry were purchased from BD bioscience Co., Ltd. (Franklin Lakes, NJ, United States). BCA, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) test kit were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China).