Separation of CD26+ cells by magnetic cell sorting (MACS)
MACS MicroBeads (Miltenyi Biotec, Germany) were used for the separation of cells expressing CD26. Lymphocytes were collected at day three after stimulation. At first, the mouse anti-human CD26 mAb (anti-CD26 mAb350 prepared in our own laboratory) was used to label the lymphocytes for 1 h at 4°C. Following two washing steps, magnetic MicroBeads labeled with anti-mouse IgG were added to the cells and incubated further for 15 min at 4°C. After a washing step, cells were loaded into the column, which was pre-placed in the magnetic field of a suitable MACS Separator (Miltenyi Biotec, Germany). The unlabeled cells were collected after flow-through with two times wash processes. The labeled CD26+ cells were bound to the column. After removing the column from the separator and placed in a suitable collection tube, the labeled CD26+ cells were separated from the column and flushed out by help of a plunger. Finally, two groups of cells, CD26 high-expressing (CD26high) group and CD26 low-expressing (CD26low) group, were obtained and then analyzed by flow cytometry.