Victor x3 microplate reader

The cytotoxicity of the active compound was tested with Vero E6 cell lines. Each cell line was seeded at 1 × 10⁴ cells per well into 96-well plates in growth medium and incubated overnight. The compounds were prepared at the concentrations of 2–500 µM in DMSO and added to the cells at the DMSO final concentrations of 0.1%. Cells were incubated for 48 h before analyzing the cell viability using CellTiter 96^® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The plate was read at the A450 by VICTORTM X3 microplate reader (PerkinElmer, Waltham, MA, USA). The EC₅₀ values of the cells were calculated from nonlinear regression analysis. The results were reported as means and standard error of mean (S.E.M.) from three biological independent experiments.

PGE₂ production was evaluated using the PGE₂ Immunoassay Kit (R&D System, Minneapolis, MN, USA). Primary mouse articular chondrocytes were seeded in 96-well plates (2 × 10⁴ cells/well). The levels of intracellular and secreted PGE₂ were measured in total cell lysates. Total collagenase activity in the conditioned medium of the articular chondrocyte culture was determined using the EnzCheck Gelatinase/Collagenase Assay Kit (Molecular Probes, Carlsbad, CA, USA) and measured with a VICTOR X3 Microplate Reader (PerkinElmer, Waltham, MA, USA) at excitation/emission wavelengths of 490/530 nm, according to the manufacturer’s protocol. The NF-κB reporter gene plasmids were transfected into mouse articular chondrocytes via Lipofectamine Plus (Invitrogen, Carls-bad, CA, USA). After incubating the transfected cells for 24 h in complete medium, lucif-erase activity was assessed using an assay kit (Promega, Madison, WI, USA) and then normalized to β-galactosidase activity.

Human embryonic kidney 293 cells (HEK 293) were used to test the effects of core promoter mutation in gene expression using the dual-luciferase reporter system. Cells were grown in Dulbecco’s modified Eagle’s media/Nutrient Mixture culture medium with 10% fetal bovine serum, 100 IU/ml penicillin and 100 IU/ml streptomycin sulfate. The wild-type and mutated core promoter sequences were synthesized, cloned into pGL3 luciferase reporter vector, and validated by Sanger sequencing (BGI TECH SOLUTIONS, Beijing, China). Fifty nicrogram of pGL3 containing the targeted core promoter sequences and 5 μg of control pRL Renilla luciferase reporter vector were mixed, and co-transfected into HEK 293 cells by using Lipofectamine 3000 Transfection Reagent (Thermo Fisher SCIENTIFIC, MS, USA). Forty-eight hours after the transfection, cells were harvested to measure luciferase activity by using the Dual-Luciferase Reporter Assay System (Promega, WI, USA) following the instruction (PerkinElmer Victor X3 Microplate Reader, OH, USA). Three independent tests were performed for each core promoter. Luciferase activity was normalized by dividing firefly luciferase activity with Renilla luciferase activity:
$$E_l=E_f/E_r$$
E_f: firefly luciferase activity, E_r: Renilla luciferase activity, E_l: normalized luciferase activity.

An ELISA kit (Enzo Life Sciences, Cat. number ADI-EKS-810A) was used to evaluate the concentration of HO-1. Firstly, 50 mg portions of skeletal muscle were cut into small pieces and homogenized in 1 mL of extraction reagent with the addition of protease inhibitors. Tissues were homogenized in glass homogenizer on ice. The homogenates were centrifuged at 21,000 ×g in 4°C for 10 min. Supernatants were removed and assayed immediately according to the manufacturer's instructions. Optical density was read at 450 nm using a Victor x3 microplate reader (Perkin Elmer, USA). All tests were performed in duplicate. Protein concentration of the samples was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, USA), according to the manufacturer's instructions. The HO-1 concentration was expressed as ng/mg protein.

TNF-α, IL-6, and SOD-1 concentrations in the skeletal muscle were assayed by specific enzyme linked immunosorbent assay using a commercially available ELISA test kit containing a monoclonal antibody specific for rat TNF-α, IL-6, and SOD-1 (Cloud-Clone Corp., USA). Firstly, 50 mg portions of skeletal muscle were cut into small pieces and homogenized in 2 mL of ice-cold PBS with a glass homogenizer on ice. The resulting suspension was subjected to two freeze-thaw cycles to further break the cell membranes. The homogenates were centrifuged for 5 min at 5000 ×g in 4°C, and the supernatants were collected and assayed immediately according to the manufacturer's instructions. Optical density at 450 nm was read using Victor x3 microplate reader (Perkin Elmer, USA). All tests were performed in duplicate. Protein concentration of the samples was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, USA), according to the manufacturer's instructions. The TNF-α and IL-6 concentrations were expressed as pg/mg protein. The concentration of SOD-1 was expressed as ng/mg protein.

Antibody production was quantified from the peripheral blood of mice obtained at 0, 15, 30, and 45 days after immunization using the indirect ELISA method [25 (link)]. For this, 96-well plates (Nunc Maxisorp, Thermo Fisher Scientific, MA, USA) were coated with 5 μg of recombinant proteins per milliliter of carbonate-bicarbonate buffer (50 mM, pH 9.6) and incubated for 16 h at 4°C in a humidity chamber. Plates were washed with TPBS buffer (PBS plus 0.05% Tween 20) and blocked with 0.8% gelatin for 1 h at 37°C in order to avoid nonspecific binding. To each well, 100 μl of serum was added in serial dilutions using TPBS plus 0.2% gelatin, starting at 1 : 200 dilutions. Samples were incubated for 3 h at 37°C. After this time, rabbit anti-mouse IgM, IgG, IgG1, or IgG2a secondary antibodies conjugated with horseradish peroxidase (Serotec, Oxford, UK) diluted 1 : 1000 were added and incubated for 45 min at 37°C. The reaction was revealed with 100 μl SigmaFast (Sigma-Aldrich, St. Louis, MO, USA) OPD. The final reaction was stopped with 50 μl of sulfuric acid 2 N and read at 490 nm using a VictorX3 microplate reader (PerkinElmer, USA). Results were expressed as mean ± standard deviation (SD) of the inverse from the last dilution reached before the cut-off. All experiments were done in triplicate.

TCs+, TCs- and clones at P4 were seeded in 24-well plates at 10⁴ cells/cm² and differentiated for 14 days with a pulsed adipogenic medium (de Girolamo et al. 2009 (link)) consisting of 3 days of induction in CM supplemented with 1 μM dexamethasone, 10 μg/mL insulin, 500 μM 3-isobutyl-1-methylxanthine and 200 μM indomethacin (all from Sigma-Aldrich), followed by 3 days of maintenance in CM supplemented with 10 μg/mL insulin. The cells were fixed in 10% neutral buffered formalin for 1 h and stained with Oil Red O (Sigma-Aldrich) for 15 min to evaluate lipid vacuoles formation. Oil Red O was unstained with 100% isopropanol and then quantified by absorbance at 490 nm (Perkin Elmer Victor X3 microplate reader; Perkin Elmer, Waltham, MA, USA).