TsoAg-Induced Lymphocyte Proliferation Assay

Every two weeks, three mice per group were euthanized to isolate spleen lymphocytes under sterile conditions. Then, 1 × 10⁵ cells in 200 μL of RPMI-1640 culture medium (CM) were cultured in each well of 96-well culture plates and stimulated with 5 μg/mL of TsoAg (containing native SAG1 and SAG2) as described previously [3 (link)]. In addition, CM-treated cultures without TsoAg stimulation were conducted and used as controls. The TsoAg-induced lymphocyte proliferation was then monitored using the BrdU (5-bromo-2′-deoxyuridine) Colorimetric Cell Proliferation ELISA Kit (Roche) according to the manufacturer’s instructions. Finally, the stimulation index (SI = OD₄₅₀ values from TsoAg-treated cultures/OD₄₅₀ values from CM-treated control cultures) of each group was calculated as described previously [3 (link), 4 (link)].