Taqman gene expresson master mix

DMEM medium, antibiotics (streptomycin and penicillin), fetal bovine serum (FBS) were purchased from PAA (Pasching, Austria). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Irisin was purchased from Cayman. Annexin V kit, Fc block antibodies, Cytofix/Cytoperm solution, PerCP-Cy5.5 streptavidin, Cytometric Bead Arrays for JNK, ERK and p38 quantification were acquired from BD Biosciences Pharmingen (San Diego, CA, USA). Phospho-NF-κB (ser536) Alexa Fluor 647 Conjugate rabbit monoclonal antibody was acquired from Cell Signaling Technology (Beverly, MA, USA). Elisa kit for HMGB1 and IL-1β were purchased from IBL International (Hamburg, Germany). RNeasy Plus Mini Kit (74134) for RNA isolation was obtained from Qiagen (Hilden, Germany). High Capacity RNA-to-cDNA Kit (4387406) for reverse transcription and TaqMan Gene Expresson Master Mix (4369026) for real time PCR were obtained from Applied Biosystems (ThermoFisher; Foster City, CA, USA).

Quantitative Real time PCR was used to assess TLR4, TNFα, IL-1β, IL-6, MCP-1, KC and HMGB1 gene expression. RNA was extracted from RAW 264.7 cells using RNeasy Plus Mini Kit (Qiagen, 74134) and reverse transcription was carried out using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, 4387406) according to the manufacturer’s protocols. Real time PCR was performed using TaqMan Gene Expresson Master Mix (Applied Biosystems, 4369026) in Step One Plus thermocycler (Applied Biosystems). The expression of analyzed genes was normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a housekeeping gene. mRNA expression of each sample was determined in four-five (separate RNA isolation) using the 2^−ΔΔCt method.