CD4+ T lymphocyte isolation by magnetic activated cell sorting (MACS)
PBMCs were obtained from the healthy people according to the manufacturer’s instructions (Hao Yang Biological Manufacture Co., Ltd., Tianjin, P.R.Chian). PBMCs were added with 160μl buffer and 40 μl MASC CD4 microbeads (130-045-101, Miltenyi biotech, Bergisch Gladbach, Germany) and incubated at 4 ℃ for 15 min. The separation was performed according to the manufacturer’s instructions. Cells were passed through a MACS column (type LS), the non-adherent cells were separated from CD4+ T cells by negative MACS selection. At least 106 CD4+ T cells were collected for later use.
Coculture ofthy0517 cells and CD4+ T lymphocytes
The full grown thy0517 cells in the culture bottle were digested and made into cell suspension. Cocultures of thy0517 cells and CD4+ T lymphocytes were performed using a six-well Transwell system (0.4-mm pore size membrane; Corning, Cambridge, MA, USA). 1×105 CD4+ T lymphocytes were added to the top portion, while equal volume of thy0517 cells were added on the bottom. Different concentrations of PHA(phytohemagglutinin) were added in the transwell top portion according to groups. Lymphocytes were harvested after 3 days, Th17 and Tregs were analyzed using flow cytometry in each well. This experiment was divided into 4 groups: Group A: CD4+ T lymphocytes; Group B: CD4+ T lymphocytes and thy0517 cells coculture; Group C: CD4+ T lymphocytes and thy0517 cells coculture and 10 μ g / ml PHA were added; Group D: CD4+ T lymphocytes and thy0517 cells coculture and 20 μ g / ml PHA were added. Repeat the above experiment for three times.