Mouse bone marrow-derived dendritic cells (BMDCs) were generated from C57BL/6 mice according to Ref. (23 (link)). At 7 day of culture, BMDCs were incubated in RPMI medium supplemented with 10% FCS, 5 µM 2-ME, 1 mM glutamine, and 1 mM sodium pyruvate for 2 h with different concentrations of free α-GalCer, fdWT bacteriophages, or fd/α-GalCer bacteriophages. The experiment OTI hybridoma cell experiment was performed by incubating BMDCs with different concentration of fdOVA, fdOVA/α-GalCer, or OVA257–264 synthetic peptide. After the incubation, cells were washed and stained with PE-conjugated anti mouse α-GalCer:CD1d complex (L363, Biolegend) or co-cultured (50,000/well) with the mouse Vα14 iNKT hybridoma FF13 or OTI hybridoma (50,000/well) for 40 h.
PE-conjugated anti-mouse α-GalCer:CD1d complex (L363, Biolegend) antibody was used to stain DCs and fluorescence of stained cells was analyzed by FACSCanto II flow-cytometer and DIVA (Data-Interpolating Variational Analysis) software (Becton Dickinson). The IL-2 released into cell co-culture supernatants was measured by ELISA. Supernatants (0.1 ml/well) were assayed in duplicate using mouse IL-2 ELISA MAX™ Standard (Biolegend), according to the manufacturer’s instructions.
PE-conjugated anti-mouse α-GalCer:CD1d complex (L363, Biolegend) antibody was used to stain DCs and fluorescence of stained cells was analyzed by FACSCanto II flow-cytometer and DIVA (Data-Interpolating Variational Analysis) software (Becton Dickinson). The IL-2 released into cell co-culture supernatants was measured by ELISA. Supernatants (0.1 ml/well) were assayed in duplicate using mouse IL-2 ELISA MAX™ Standard (Biolegend), according to the manufacturer’s instructions.