Poly 1 c

For induction of Cre-mediated deletion of floxed gene alleles in Mx1-Cre S1P reporter mice, 3- to 5-day-old mice received a single intraperitoneal injection of 70 μl polyI:C (GE Healthcare) at a concentration of 2 mg/ml in PBS. All mice in each litter were treated identically. For induction of Mx1-Cre in Cxcr4^f/fMx1-Cre mice, adult mice received intraperitoneal injections of 100 μl polyI:C at a concentration of 2 mg/ml in PBS once every other day for a total of 3 injections. LNs were harvested at least at least 9 days after the last treatment. For induction of UBC-CreERT2, adult mice received daily intraperitoneal injections of 200 μg tamoxifen for 5 days, and mice were analyzed at least 5 days after the final tamoxifen dose.
NIBR-0213, provided by Novartis, was used to inhibit S1PR1 internalization in S1P reporter mice. Dessicated powder was resuspended at 3 mg/ml in 30% polyethylene glycol 200 (TCI America) in PBS^{36 (link)}. Mixture was heated to 45° C for 1 h to dissolve the powder. Animals were injected once daily with 250 μl (30 mg/kg) for 8 days. LNs were collected 3–5 h after the last treatment.
FTY720 (Cayman Chemical) was used to induce S1PR1 internalization in S1P reporter mice. Mice were treated with FTY720 at a dose of 1 mg per kg body weight (mg/kg) intraperitoneally 5 h before harvest of LNs.

For 6-hour immunizations, 2 × 10⁸ SRBCs (Colorado Serum Company), 20 μg of LPS (0111:B4, Sigma), 150 μg of poly I:C (GE Healthcare), or saline was injected retro-orbitally into mice. For immunization of mice with both SRBC and poly I:C, SRBCs were resuspended directly in corresponding doses of poly I:C.
For analysis of CD4⁺ T cell responses, pooled spleen and LN cells containing 10⁶CD45.1⁺CD4⁺Vα2⁺ OTII were adoptively transferred into mice. For analysis of B cell responses, spleen cells containing 10⁵ HEL-binding Hy10 B cells and 10⁴ OTII T cells were cotransferred. One day after cell transfer, recipients were immunized intraperitoneally with HEL-OVA conjugated to SRBCs (SRBC-HEL-OVA; 2 × 10⁸ cells).