Live cells were detected by trypan
blue dye exclusion, and the leukocyte concentrations were
adjusted to 1 107 cells/mL in RPMI 1640 with 10% fetal bovine
serum. A 190-lL cell suspension was co-incubated with Con A
(Sigma, final concentration of 35 lg/mL) and lipopolysaccharide
(Sigma, final concentration of 20 lg/mL) in 96-well plates at a total
culture volume of 200 lL. The cells were then incubated at 41 C in
a humidified atmosphere of 5% CO2 for 68 h.