*5.8. Evaluation of IL-17 and TNF-α Release by Multiplex Assay*

To investigate the IL-17 and TNF-α cytokines release, EAE animals treated with CRO or saline were euthanized, and the lumbar portion of the spinal cord (L3-L6) was collected on the 17th day after immunization. Collected tissue was homogenized in RIPA buffer (Sigma-Aldrich™, USA) containing protease inhibitors cocktail (1:100, Sigma-Aldrich™, USA) and phosphatase (1:300, Sigma-Aldrich™, USA). The homogenate was centrifuged for 5 min at 10,000× *g* and 4 ◦C. An aliquot of the supernatant was used for protein determination by the Bradford method [39,101]. The samples were normalized (3 μg/μL) and the protein concentration was determined using the commercial kit (Millipore, Burlington, MA, USA) by xMap method (MULTIPLEX), All the samples were done in duplicate and read using the Luminex 200 equipment—xPonent software version 4.2 (LEAC lab, Sao Paulo, SP, Brazil).
