Fix buffer

AnnexinV-FITC/PI Kit (KeyGen Biotechnology, Nanjing, China) was used to evaluate the effect of FICZ and ITE on the apoptosis of PBMCs. For analysis of the frequency of Th1 and Th17, the cells were stimulated by adding PMA (50 ng/mL, Sigma-Aldrich, St. Louis, MO) and ionomycin (1 ug/mL, Sigma) for 1 h at 37°C. Then, brefeldin A (10 ug/mL, Sigma) was added for another 4 h; the cells were fixed and permeabilized using the eBioscience Cytofix/Cytoperm kit according to the manufacturer's instructions and then incubated with CD3-(PerCP)-Cy5.5, anti-human CD8-APC, anti-human IL-17A-PE, and anti-human IFN-γ-FITC (BD Biosciences). For phosphorylated STAT staining, stimulated CD4⁺T cells were fixed with Fix buffer (BD Biosciences) for 10 min at 37°C, permeabilized with Perm buffer (BD Biosciences) for 30 min on ice, and stained with anti-human pSTAT3-PE, anti-human pSTAT4-Per-cy5.5, anti-human pSTAT4-Per-cy5.5, anti-human pSTAT5-PE, or isotype control mAbs (BD Biosciences). Flow cytometric analysis was performed on a FACScan flow cytometer (BD Biosciences) to measure mean fluorescence intensity (MFI). Results were expressed as the percentage difference compared with isotypic control (IC) using the formula [mean fluorescence intensity (MFI) of sample − MFI of IC]/MFI of IC.

Confluent PK-15, PoREC, IPEC-J2 cells were treated with 300ng/ml IFN-α or IFN-λ3 for 24h, incubated for 20 min at 37°C with accutase and collected in 96-well V-bottom plates. Cells were washed two times in PBS between each step. Cells were fixed with Fix buffer (BD Biosciences) for 20min at 4°C as positive control for propidium iodide (PI) staining, which was used to measure cell viability. Flow cytometry was performed using a NovoCyte Flow Cytometer (ACEA Biosciences, Agilent, Santa Carla, CA, USA), and samples were analyzed with NovoExpress software (ACEA Biosciences).