For immunization studies, mice were subcutaneously immunized with 50 µg KLH (Sigma-Aldrich) or 100 µg NP-OVA (Biosearch Technologies) emulsified in CFA (Sigma-Aldrich), and lymphoid cells from the draining LNs were stained and analyzed by flow cytometry on day 8 to 10. For viral infection experiments, mice were intraperitoneally injected with LCMV-Armstrong (2 × 105 pfu), and lymphoid cells in the spleen were analyzed on day 8. In some experiments, mice were intraperitoneally treated with either Dimethyl sulfoxide (DMSO) vehicle or 20 mg/kg GW3965 (Tocris).
For BM chimera studies, 8- to 10-wk-old sublethally-irradiated Rag1−/− mice (9 Gy; X-RAD IR160, Precision X-Ray, USA) were i.v. injected 1:1 mixture of WT and Nr1h2−/− BM cells before 6 wk of reconstitution. The recipients were s.c. immunized with KLH in CFA or infected with LCMV, and lymphoid cells from the draining LNs or spleen were analyzed as indicated (46 (link)).
For OT-II T cell cotransfer studies, 1:1 mixture of flow-sorted WT and Nr1h2−/− naïve OT-II T cells (2 × 106 cells) were i.v. transferred into sex-matched B6.SJL congenic recipient mice. One day later, the recipients were s.c. injected with 100 µg OVA (Sigma-Aldrich) emulsified in CFA and lymphoid cells from the draining LNs were analyzed on day 8.
For BM chimera studies, 8- to 10-wk-old sublethally-irradiated Rag1−/− mice (9 Gy; X-RAD IR160, Precision X-Ray, USA) were i.v. injected 1:1 mixture of WT and Nr1h2−/− BM cells before 6 wk of reconstitution. The recipients were s.c. immunized with KLH in CFA or infected with LCMV, and lymphoid cells from the draining LNs or spleen were analyzed as indicated (46 (link)).
For OT-II T cell cotransfer studies, 1:1 mixture of flow-sorted WT and Nr1h2−/− naïve OT-II T cells (2 × 106 cells) were i.v. transferred into sex-matched B6.SJL congenic recipient mice. One day later, the recipients were s.c. injected with 100 µg OVA (Sigma-Aldrich) emulsified in CFA and lymphoid cells from the draining LNs were analyzed on day 8.
