Splenocytes from C57BL/6 or BALB/c mice were exposed to 3000 rad of gamma radiation and used as stimulator cells. The graft recipient splenocytes were then incubated in triplicate cultures with an equal number of C57BL/6 stimulator cells (experimental group), BALB/c stimulator cells (negative control), or BALB/c stimulator cells plus 0.1 μg/mL of anti-CD3 (Clone 145-2C11; BD Pharmigen, Franklin Lakes, NJ, USA) as a positive control. After 3 days in culture, the supernatants and the cells were harvested.
The supernatants were analyzed by sandwich ELISA for IFN-γ. Briefly, Immulon 4HBX high binding polystyrene ELSIA plates (Thermo-Fischer, Rochester, NY, USA) were coated with anti–IFN-γ (clone R4-6A2) over night at 4°C, washed with PBS + 0.05% tween 20 (Sigma-Aldrich Corp., St. Louis, MO, USA), and the wells were blocked for 1 hour with PBS + 0.05% tween and 1% BSA (Sigma-Aldrich Corp.). The wells were washed and then loaded with either 100 μL of either diluted supernatant from the lymphocyte cultures, or of various concentrations of a purified mouse IFN-γ standard. The plates were incubated overnight at 4°C, the wells were washed and loaded with biotin conjugated anti–IFN-γ (clone XMG1.2; BD Pharmigen). After 1 hour at room temperature the wells were washed, loaded with streptavidin conjugated to horseradish peroxidase, and incubated for 30 minutes at room temperature. The wells were washed and loaded with 100-μL TMB peroxidase substrate (Life Technologies, Fredrick, MD, USA). Color development was stopped by addition of 50 μL of 1N H2S04, and absorbance was read at 450 nm using a Synergy 2 plate reader (BioTek, Winooski, VT, USA).