MHC-1 Stabilization Assay

RMA-S cells were infected with the VSV.G plasmid expressing the 97W or 97R version of L^d and sorted for L^d-positive cells using the Thy1.1 marker. The MHC-1 stabilization assay was performed as described (64). In brief, RMA-S.L^d (97W or 97R) cells or splenocytes from L^d 97W and 97R mice were incubated at room temperature overnight to increase surface MHC-1 expression. The next day, cells were washed with PBS and plated at 3 × 10⁵ cells per well in a 96-well round-bottom tissue culture plate. Peptides of interest (Peptide 2.0 Inc.) were added to the cells at indicated concentrations. The cells were incubated for 1 h at room temperature and 3 h at 37°C. Cells were stained with the 30-5-7 antibody (specific for conformed, peptide-bound L^d) and a goat anti-mouse IgG phycoerythrin (PE)-conjugated secondary antibody and analyzed by flow cytometry.