Evaluation of SMI Effects on Murine T Cells

According to the literature, the spleen of the mice was used to prepare single-cell suspension (18 (link)). Then, the effects of SMI on mice T helper (Th1, Th2, Th17) cells and regulatory T (Treg) cells were determined using flow cytometry. In brief, the spleen of mice was digested and single-cell suspensions were stimulated with 50 ng/mL PMA and 1 μg/mL ionomycin in the presence of BD GolgiPlug (Leuko Act Cktl with GolgiPlug, BD Biosciences, San Jose, CA, USA) for 10 hours. Cells were fixed and permeabilized using BD Transcription Factor Buffer Set (BD Biosciences, San Jose, CA, USA) followed by staining with fluorescence-labeled antibodies CD3e-BV605 (145-2C11), CD4-APC-H7 (GK1.5), CD8a-BV510 (53-6.7), CD25-BV421 (PC61), FoxP3-Alexa 647 (MF23), IFN -γ-FITC (XMG1.2), IL-4-PE-Cy7 (11B11) and IL-17A-PE (TC11-18H10). All fluorescence-labeled monoclonal antibodies were purchased from BD Pharmingen. Flow cytometry was performed on a Beckman Coulter CytoFlex flow cytometry system.