β-Galactosidase X-Gal and ONPG assays.

Cultures containing a mixture of B3Z T cell hybrids and DC2.4 APCs (2 × 10⁵ cells/ml each) were plated in 6-well tissue culture plates (X-Gal assays) or 96-well flat-bottom tissue culture plates (ONPG assays), and peptides or bacteria were added. Peptides (listed in Table 2) were synthesized and purchased from GenScript and resuspended in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mg/ml. B. melitensis and variants were used at an MOI of 100. For some assays, APCs were treated with tauroursodeoxycholic acid (TUDCA; Sigma) at 100 μg/ml, or tunicamycin (Sigma) at 10 μg/ml, or mouse IFN-γ (PromoKine) at 1 μg/ml. After overnight incubation, cells were washed in PBS and fixed (X-Gal assay) or lysed (ONPG assay). For X-Gal staining, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, washed 3 timed in PBS, and overlaid with a solution of 1 mg X-Gal/ml, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl₂. After an overnight incubation at 37°C, plates were examined microscopically for the presence of blue (lacZ expressing) cells. For ONPG staining, we used a SensoLyte ONPG β-galactosidase assay kit (AnaSpec, Inc.) as per the manufacturer's recommended protocol, except that the incubation at 37°C was overnight. Absorbance reading was at 405 nm.