2.9. Splenocyte Proliferation Assay

The spleens that were isolated from each group of mice were gently homogenized with PBS and passed through a 200-mesh filter to obtain a homogeneous splenocyte suspension. The erythrocytes were removed from the suspension using erythrocyte lysis buffer, and the remaining splenocytes were then suspended in complete RPMI-1640 media that contained 10% FBS, 100 μ/mL penicillin, and 100 μg/mL streptomycin. Then, 2.0 × 10⁵ purified splenocytes were seeded into each well of a 96-well microplate and mixed with Con A (5 μg/mL) or LPS (10 μg/mL) in 200 μL volume. The plates were then incubated at 37^oC in a 5% CO₂ humidified incubator for 48 h. Finally, the cellular proliferation was determined using a CCK-8 kit, as described in the literature [14].