T cell hybridomas were stimulated by the indicated peptides presented by primary macrophages. T cell clones restricted by H2-A
b (H5, H18 and OT-II) were stimulated by C57BL/6J-derived macrophages, whereas the H2-A
d-restricted clone (DO11.10) was stimulated by BALB/cJ-derived macrophages. Similar results were also obtained when primary splenic B cells or bone-marrow-derived dendritic cells were used for presentation. Approximately, 1.5×10
5 T cells per well were used in flat-bottomed 96 well-plates in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal calf serum. The following day, T cell activation was assessed by flow cytometric detection of CD69 induction, using a fluorescein isothiocyanate-conjugated monoclonal anti-mouse CD69 antibody (armenian hamster; clone H1.2F3; eBioscience, San Diego, CA, USA; cat. no. 11-0691-85) at 1 in 200 dilution. At least 50,000 cells were acquired on LSRFortessa X-20 or FACSCanto II cytometers (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo v10 (Tree Star Inc., Ashland, OR, USA). Unstimulated hybridoma cells were included as CD69-negative controls, based on which the gating of CD69-postitive cells was drawn.
b (H5, H18 and OT-II) were stimulated by C57BL/6J-derived macrophages, whereas the H2-A
d-restricted clone (DO11.10) was stimulated by BALB/cJ-derived macrophages. Similar results were also obtained when primary splenic B cells or bone-marrow-derived dendritic cells were used for presentation. Approximately, 1.5×10
5 T cells per well were used in flat-bottomed 96 well-plates in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal calf serum. The following day, T cell activation was assessed by flow cytometric detection of CD69 induction, using a fluorescein isothiocyanate-conjugated monoclonal anti-mouse CD69 antibody (armenian hamster; clone H1.2F3; eBioscience, San Diego, CA, USA; cat. no. 11-0691-85) at 1 in 200 dilution. At least 50,000 cells were acquired on LSRFortessa X-20 or FACSCanto II cytometers (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo v10 (Tree Star Inc., Ashland, OR, USA). Unstimulated hybridoma cells were included as CD69-negative controls, based on which the gating of CD69-postitive cells was drawn.