The cells collected at the interface werewashed
with PBS, and lymphocyte separation was repeated to achieve a
lower degree of contamination by erythrocytes. After washing the
cells with RPMI containing 2 mM L-glutamine (Sigma), the cells
were counted by a cell counter (Coulter, Fullerton, CA, USA) and
resuspended with RPMI1640 supplemented with gentamicin
(150 mg/mL), 10% fetal calf serum, and L-glutamine (2 mM) at a
working concentration of 2 106 cells/mL.