2.4 Determination of Lymphocyte Proliferation Activity

2.4 Determination of Lymphocyte Proliferation Activity

Preparation of Splenic Cell Suspension: The spleen was aseptically harvested and rinsed in PBS solution. It was then gently crushed between two slides and filtered through a 200-mesh nylon cloth to obtain a single-cell suspension [23]. Next, lymphocytes were isolated using lymphocyte separation solution [24], and the cell concentration was adjusted to 1×10⁷ cells/mL [25].

Determination of Lymphocyte Proliferation Activity: The above-prepared splenic lymphocyte suspension (1×10⁶ cells/mL) was added to a 96-well plate, 100 μL per well, with four replicates. Two wells were supplemented with 100 μL of 1640 culture medium containing ConA (final ConA concentration: 7.5 μg/mL) [26], while the other two wells received 100 μL of 1640 culture medium alone. The plates were then incubated at 37°C with 5% CO₂ for 72 hours. Six hours before the end of incubation, 15 μL of MTT solution (5 mg/mL) was added to each well, and the incubation continued. After incubation, the plates were centrifuged at 2000 rpm for 10 minutes at room temperature, the supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were shaken to ensure complete dissolution of the crystals, and the A₄₉₀ value was measured using a microplate reader [27].