At 8-week post infection (p.i.), one-half of the spleen from each euthanized mouse was used to produce single cell suspensions as previously described. The cell density was adjusted to 2 × 10 6 per ml using complete RPMI-1640 containing 10% (v/v) heatinactivated FBS, 1 mM non-essential amino acid, 100 μg/ml of penicillin and 100 U/ml streptomycin. The cell suspension was dispensed to 24-well plates with 1 ml/well and stimulated with heat killed Brucella (HKB) or ConA (2 μg/ml) for three days. Following stimulation, supernatants were collected and cytokine synthesis characterized. Cells collected from 100 μl of the suspension were lysed using 1% (v/v) Triton X-100, and the LDH released from live cells was determined using CytoTox 96™ Nonradioactive Cytotoxicity Assay kit (Promega) following the manufacturer's instructions.