Milliplex rat cytokine chemokine magnetic bead panel kit

Twenty-one days after administration of DSP-4 (n=8) or saline (n=8), fresh spinal lumbar enlargements (L4–L6) were collected and diluted in lysis buffer (20mM TRIS pH 7.4, 150mM NaCl, 2.5% glycerol, 1% Triton X-100, 1% Chaps, 1mM EDTA, 1mM EGTA, 0.1% SDS, 1% Halt protease and phosphatase inhibitor cocktail, 2mM PMSF) with a 1:10 w/v ratio. Tissues were sonicated using a probe sonicator. Lysates were centrifuged (13,000×g, 15min) at 4°C and supernatants were collected and stored at −20°C. Protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). A Milliplex Magnetic Bead Panel kit (Milliplex Rat Cytokine/Chemokine magnetic bead panel kit, EMD Millipore, Billerica, MA) was used to quantify the expression levels of TNF-α, IL-1β, IL-6, IL-10, IL-17A, and IFNγ. The beads were evaluated in a Luminex 200 instrument, and the data were collected and analyzed using Milliplex Analyst software (Millipore, Billerica, MA, USA). A minimum of 50 beads were analyzed. Results were expressed as picograms per milligram of wet tissue (pg/mg) using independent sampling.