Quantikine immunoassay kit

Human bone marrow-mesenchymal stem cells (hBM-MSCs) were purchased from Lonza, Inc. (Walkersville, MD, USA) and cultured in low-glucose (1 g/L) DMEM supplemented with 10% FBS, penicillin-streptomycin, and Glutamax^TM (Invitrogen, Carlsbad, CA, USA). To induce adipogenesis, the cell growth medium was replaced with high-glucose (4.5 g/L) DMEM supplemented with 10% FBS, penicillin-streptomycin, 10 μg/mL insulin, 0.5 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (IDX conditions) [30 (link)]. IBMX, pioglitazone, and aspirin were purchased from Sigma-Aldrich (St. Louis, MO, USA). hBM-MSCs were stained with 0.2% oil red O (ORO) reagent for 10 min at 24 °C, and then washed with H₂O four times. Following a 10-min elution with isopropanol, the absorbance was measured at 500 nm using a spectrophotometer. To visualize the nucleus, the hBM-MSCs were counterstained with hematoxylin reagent for 2 min and then washed twice with H₂O. The level of adipocyte differentiation was observed and counted using an inverted phase microscope. A Quantikine immunoassay kit (R&D Systems, Minneapolis, MN, USA) was used for quantitative determination of adiponectin in the cell culture supernatants.

Jurkat cells harvested after 48 hours were centrifuged at 200 ×g for 10 min to collect cells and culture medium. Cells were lysed in 50 mM Tris-HCl (pH 7.4), containing protease inhibitors, and cytokine content was quantified by coating proteins (20 μg/well) from whole lysates overnight in a 96-well ELISA microplate, as reported [40 (link)]. Rabbit anti-LIF, anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-10, and anti-INFγ (diluted 1 : 500) were used as primary antibodies; GAR-AP (diluted 1 : 2000) was used as secondary antibody and absorbance values were read at 405 nm. Release of LIF and other cytokines from Jurkat cells into the medium was quantified through Quantikine Immunoassay kit (R&D System, Minneapolis, MN, USA) and a specific Multiprotein Profiling ELISA Kit (SuperArray Bioscience Co., Germany), respectively, according to the manufacturer's instructions. To this aim, 50 μL of culture medium was used, and the content of each protein was evaluated by comparing A_405 nm values to those of antigen standard curves (positive controls).

MCP1 and CSF1 protein levels were determined by ELISA using the Quantikine Immunoassay kit (R&D Systems, Minneapolis, MN) or by Milliplex MAP analyses Millipore, MA) according to the manufacturer's protocol. Samples were assayed in duplicate, and data represent the mean concentration average over triplicate experiments.

Quantitative levels of murine IL-11, CTGF, VEGF, PTHrP, and TGF-β1 in serum, bone marrow isolated from mice and the conditioned media of cultured cells was determined in triplicate by ELISA according to the manufacturer’s protocol (Quantikine immunoassay kit, R&D systems).

Glucose content and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in the serum, were measured using the blood biochemical analyzer (Fuji Dri-Chem 3500). The levels of leptin and adiponectin in the serum secreted by adipose tissue were measured while using a leptin mouse/rat enzyme immunoassay (EIA) kit (Quantikine & Immuno Assay kit, R&D Systems, Minneapolis, MN, USA) and the adiponectin rat EIA (ALPCO Diagnostics, Salem, NH, USA), respectively, based on a sandwich-type enzyme-linked immunosorbent assay (ELISA), and then analyzed at 450 nm using a plate reader (Spectra Max 250; Molecular Devices, San Jose, CA, USA). Serum insulin levels were measured using an insulin radioimmunoassay kit (Eiken Chemical Co., Ltd., Tokyo, Japan).