Spleens were macerated, washed in Dulbecco's solution containing 2% FCS, the red blood cells lysed with 0.09% NH 4 Cl for 5 min at 37uC, and then washed again. The splenocytes were resuspended in complete DMEM containing 10% FCS, 100 U/ml of Penicillin, 100 mg/ml of Streptomycin, 2 mM L-Glutamine and 0.05 mM ß-mercaptoethanol, and cultured in 96-wells flat-bottomed plates for ELIspot or CBA at a concentration of 5610 5 cells/well.