Level of cytokines in peripheral blood was tested before and after treatments. Peripheral blood was collected in procoagulant tube and centrifuged in 3500 rpm/min for 5 minutes. Serum was then transferred into EP tubes. Each tube contained 200 microliters of serum and was saved in −80°C freezer for further testing. BD Multitest_IMK kit was used to detect the level of TNF-α and IL-6 in serum, and multifunction streaming LUMINEX 200 was used for analysis and detection.
Regulatory T cells (Tregs) were stained with anti-CD4 (BD, number 340133) fluorescein isothiocyanate, anti-CD25-Allophycocyanin (BD, number 340939), and anti-Foxp3-PE (eBioscience, number 12-4776-42). Isotype control antibodies were used alternatively. Samples were incubated in the dark for 15 minutes and analyzed with flow cytometer (BD, FACS Calibur, USA). Image and data were acquired and saved.
Regulatory T cells (Tregs) were stained with anti-CD4 (BD, number 340133) fluorescein isothiocyanate, anti-CD25-Allophycocyanin (BD, number 340939), and anti-Foxp3-PE (eBioscience, number 12-4776-42). Isotype control antibodies were used alternatively. Samples were incubated in the dark for 15 minutes and analyzed with flow cytometer (BD, FACS Calibur, USA). Image and data were acquired and saved.