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.