4.4. Lymphocyte Proliferation Assay

Splenocytes were cultured under the general cell culture condition [19] and incubated in the presence of a mitogen such as Con A (1 µg/mL) or lipopolysaccharide (LPS; 100 ng/mL) and their proliferative activity was assessed using a ³(H)-thymidine incorporation assay. A solution containing 1 µCi of ³(H)-thymidine was added to each well and incubated for an additional 16 h until the total incubation period was 72 h. Then, the cultured cells were harvested and transferred onto a glass filter, which was placed in a sample bag containing scintillation cocktail. The level of ³(H)-thymidine incorporated was measured using a microbeta counter (Wallac, Waltham, MA, USA).