Interleukin 6 (il 6)

Serum lipid profiles and fasting blood glucose (Pars Azmoon Kit) were measured by using an auto-analyzer (alpha-classical, Isfahan, Iran). The enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of fasting insulin (Monobind, Lake Forest, CA, USA. Cat# 5825-300A), TNF-α (Diaclone, Besancon, France. Cat# 950.090.096) and IL-6 (Diaclone, Besancon, France. Cat# 950.030.096). Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated by the following equation: (fasting insulin (IU/ml) × fasting glucose (mmol/l)/22.5) [26 (link)].

Studies of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) production were completed with ELISA assay. Condition media from the 3D samples were collected and stored at −70°C until the assay. Secreted TNF-α, IL-1β (both from Eastbiopharm, China) and IL-6 (Diaclone, France) were quantified according to the manufacturer's recipes.

Studies of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) production were completed with ELISA assay. Condition media from the 3D samples were collected and stored at −70°C until the assay. Secreted TNF-α, IL-1β (both from Eastbiopharm, China) and IL-6 (Diaclone, France) were quantified according to the manufacturer's recipes.

Five ml of fasting blood sample was collected, and serum was separated for estimation of all biochemical parameters following the procedures as adopted earlier^{18 (link)}. Blood glucose was estimated by enzymatic method (Randox, India). Insulin was measured using ELISA kit (Calbiotech, USA). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the formula blood glucose (mg/dL) × insulin (µIU/mL)/405. HbA1c was determined from whole blood using commercial kits for turbidimetric immunoassay (Quantia, Tulip diagnostics). Lipid parameters such as total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-c), low-density lipoprotein-cholesterol (LDL-c) were estimated using reagent method (Randox, India). Very low-density lipoprotein-cholesterol (VLDL-c) was calculated using the Friedwald’s formula (TG/5). Various lipid-risk factors were assessed and atherogenic index of plasma (AIP) was calculated by log₁₀ of TG/HDL-c. Malondialdehyde (MDA) was measured by colorimetric assay-kit (Elabscience, USA), and high-sensitive C-reactive protein (hsCRP) (Calbiotech, USA), and interleukin-6 (IL-6) (Diaclone, France) were measured using ELISA kit, according to manufacturer instructions. Nitric oxide derivatives (nitrate and nitrite) were estimated by colorimetric method using microplate reader (Elabscience, USA).

The medium of the hMADS cells was collected over 24 h, from day 13 (after replacement of medium) to day 14 of differentiation, to determine the secretion of adipokines using high-sensitive ELISAs (leptin and DPP-4 from R&D Systems, Inc., Minneapolis, MN, USA; IL-6 and MCP-1 from Diaclone SAS, Besancon Cedex, France; adiponectin and PAI-1 from BioVendor–Laboratorni medicina a.s., Brno, Czech Republic). If necessary, samples were diluted with the dilution buffer provided by the manufacturer prior to the assay, which was performed in duplicates according to the manufacturer’s instructions.

The supernatants obtained from the murine peritoneal macrophage and murine splenocyte culture were collected 18 or 24 h after LPS stimulation. Subsequently, they were processed by ELISA kits to determine levels of IL-1β (BD OptEIA^®, San Jose, CA, USA), IL-6 (Diaclone^®, Besacon Cedex, France), IL-17 and TNF-α (Peprotech^®, London, UK), according to the manufacturer’s instructions. Results were expressed in pg/mL.