Mab406

Intrathecal injections were performed for 3 consecutive days (days 7 to 9) after intra-articular injection of CFA in a subset of P21 animals. Recombinant rat anti-IL6 antibody (MAB406, R&D Systems) was reconstituted in sterile saline and diluted to the following concentrations: 3 ng/10 µL, 10 ng/10 µL and 30 ng/10 µL. Under brief isoflurane anaesthesia (3%), a 1 mL insulin syringe was inserted between the L5 and L6 spinal vertebrae and 10 µL of the desired concentration of anti-IL6 was administered. Control animals received intrathecal injections of sterile saline (10 µL). Intrathecal injections were given near the end of the light cycle of animals, after the completion of sensory behaviour tests.

For N‐formyl peptide culture, the cerebral vessels from young (2–3 months old) WT mice were dissected, harvested, and washed in PBS. Cerebral vessels from left and right hemisphere were kept separate from each mouse brain and were randomized for 30‐minute culture in PBS with either 10 μmol/L N‐formyl peptide (N‐Formyl‐Nle‐Leu‐Phe‐Nle‐Tyr‐Lys; Genscript catalog No. RP12959) or vehicle (PBS+1% dimethyl sulfoxide), similar to what was previously done.¹⁵ After 30‐minute culture, vessels were washed twice in PBS and immediately flash frozen for immunoblot analysis. For recombinant IL‐6 culture, the cerebral vessels from young (2–3 months old) mice were dissected, harvested, and washed in PBS. For recombinant IL‐6 culture, cerebral vessels were cultured in DMEM+10% fetal bovine serum with either 0 or 10 ng/mL of recombinant IL‐6 (PeproTech, catalog No. 216‐16) or vehicle for 2 hours. After 2 hours of IL‐6 culture, the tissue was washed 3× with PBS, flash frozen, and used for immunoblot analysis. For anti–IL‐6 culture, cerebral vessels were cultured in DMEM+10% fetal bovine serum with either 5 μg/mL anti–IL‐6 antibody or IgG control (R&D Systems, catalog No. MAB406, clone MP5‐20F3) for 2 hours. After 2‐hour culture, tissue was washed 3× with PBS and flash frozen for use immediately for respirometry studies.