Mixed lymphocyte reaction (MLR)

The mouse spleen lymphocytes were separated using the Ficoll method (16). The spleen was separated aseptically, rinsed repeatedly using ice-cold PBS (30 ml/wash) with a syringe, resuspended in 3 ml of PBS and slowly added to 3 ml of mouse lymphocytes in the upper separation medium. The samples were then incubated for 20 min at room temperature. The samples were centrifuged for 10 min at 2,000 × g, and the white rete in middle layer was collected, rinsed twice with PBS, and resuspended in RPMI-1640 complete culture solution. C57BL/6 lymphocytes were used to stimulate the cells, and normal BALB/c lymphocytes were used in the sensitization model as responder cells. The stimulated cells were treated with mitomycin C (25 µg/ml; Kyowa Hakko Kogyo Bio Co., Ltd., Tokyo, Japan), incubated at 37°C for 30 min, and rinsed 3 times with nutrient solution (RPMI-1640) The cell suspension (1×106 cells/ml) was prepared in RPMI-1640 nutrient solution, and 100 µl of this suspension was placed in each well of a 96-well culture plate. The responder cells were then added at a concentration of 1×105 cells in 100 µl/well. The stimulating and responding cells were also placed in separate wells for analysis (resulting in 3 wells) and cultivated at 37°C in a CO₂ (5%) incubator for 5 days. BrdU (EMD Millipore) was added to the cells, followed by an additional incubation at 37°C for 16 h. Stop buffer (Gibco; Thermo Fisher Scientific, Inc.) was added, and then the optical density of each well was tested using an ultraviolet spectrophotometer at a wavelength of 450/550 nm.