Ipsilateral popliteal lymph nodes were harvested at day 3 and day 8 after CFA injection, dissociated using a bullet blender (Next Advance) with SSB05 beads (Next Advance) in RLT buffer (Qiagen, Toronto, Ontario, Canada). Total RNA was extracted using an RNeasy Mini kit (Qiagen), according to the manufacturer’s instructions. The quality and quantity of RNA were determined using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Montréal, Quebec, Canada). Relative Proenkephalin (PENK) gene expression [normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT)] was determined by quantitative PCR using Quantitect SYBR Green PCR Master Mix (Qiagen) and a StepOnePlus real-time PCR detection system (Applied Biosystems, Burlington, Ontario, Canada). The following primers were used: 5′-GTTCTTTGCTGACCTGCTGGAT-3′ and 5′-CCCCGTTGACTGATCATTACAG-3′ for HPRT, 5′-CGACATCAATTTCCTGGCGT-3′ and 5′-AGATCCTTGCAGGTCTCCCA-3′ for PENK.

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