Elisa plate reader

Receptor mediated signaling was assessed by Luciferase reporter assay following the method of Fortin et al. with modifications46 (link). In brief, the cells were grown in 70 mm dish until they attain 70% confluence. A cAMP responsive element-luciferase reporter plasmid (CRE_6X-luc), which is a kind gift of Prof Richard Day at Indiana University, and beta galactosidase plasmid were transfected in 1:1 ratio using Turbofect transfection reagent following manufacturer’s protocol. Soluble cholesterol treatment at indicated concentrations was carried out for different time period in complete media 48 hours after the transfection following which the cells were treated with100 nM Exendin-4 for 4 h. The media was then aspirated, cells were lysed and luciferase activity was measured using Steady lite plus reagent (Perkin Elmer Life and Analytical Science, Waltham, MA). To enable the correction for the inter-well variability of transfection, beta galactosidase assay was performed by the addition of 2-nitrophenyl-beta galacto pyranoside (Sigma). After incubation for 15 min at 37 °C, substrate cleavage was quantified by measuring the optical density at 405 nm in ELISA plate reader (Perkin Elmer, USA) and the corresponding values were used to normalize luciferase activity.

Intracellular tyrosinase activity was measured using a previously described method [13 (link)] with slight modifications. Melanoblasts were seeded at 1 × 10⁴ cells/well in 6-well plates and cultured for 3 days in differentiation media. Then, the medium was exchanged for M254 containing 0.24 μg/mL OSA and 30 μg/mL RBE and the differentiated melanocytes were cultured for further 3 days. After removing the culture media from the plates, the cells were washed with PBS and lysed with 10% triton X-100 (Sigma-Aldrich). The cells were centrifuged at 15000 rpm for 15 min, and then the protein content of the supernatants was measured by the bicinchoninic assay (BCA) (Thermo Fisher Scientific, USA). 3,4,-Dihydroxyphenylalanine (L-DOPA) (Sigma-Aldrich) (10%) was dissolved in sodium phosphate buffer (10 mM). After 30 min incubation at 37°C, absorbance was measured at 475 nm using an ELISA plate reader (PerkinElmer Life Sciences).

The effect of zerumbone on tumor cell viability was assessed by using the XTT: (sodium 3′-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) cell proliferation kit (Roche Applied Science, Indianapolis, IN) as described previously.25 (link) Briefly, cells (3 × 10⁴/mL) were seeded in 96-well plates and grown overnight. The next day, the medium was aspirated and cells were exposed to different concentrations of zerumbone for 7 h. Next, the zerumbone was aspirated and the wells were rinsed and replenished with fresh medium. After a further 48 h of culture, XTT labeling mixture was added to the cells and incubated for another 4 h. The resulting formazan product was then spectrophotometrically quantified (490 nm) by using an enzyme-linked immunosorbent assay (ELISA) plate reader (Perkin Elmer, Waltham, MA). Results are expressed as percent cell viability for each concentration of zerumbone with respect to untreated controls (0.1% DMSO in medium).

The cytotoxicity of the MOL extract and its derived fractions were determined using the methyl thiazol tetrazolium (MTT) assay. MDA-MB-231 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and treated with MOL extract at concentration 75, 100, and 150 µg/mL for 24 h. MTT salt solution was added and the plate was incubated for 3 h at 37 °C. Then, formazan crystals were dissolved in 100 µl DMSO. The absorbance was measured at 570 nm using an ELISA plate reader (PerkinElmer, Inc., Waltham, MA, USA).

The decorin levels in the archived BM plasma samples were measured using DuoSet enzyme-linked immunosorbent assay (ELISA) kits (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. In brief, 96-well plates were coated with 2 μg/mL of capture antibodies per well and incubated overnight at room temperature (RT). In addition, 1% bovine serum albumin (Bionovas, Toronto, Canada) in PBS (200 μL/well) was added to block nonspecific binding. Then, 100 μL of plasma sample (diluted if indicated) was added to each well and incubated overnight at 4°C. A detection antibody (200 μL) was added and incubated at RT for 2 hours. Subsequently, the wells were incubated with a streptavidin-HRP conjugated antibody for 20 minutes followed by 100 μL of substrate solutions for 20 minutes, and then with a stopping solution. The wells were then placed on an ELISA plate reader (Perkin Elmer, CA, USA), and the absorbance at 450 nm of each well was recorded. Serial dilutions (0–800 ng/μL) of purified decorin protein were used as standards in each experiment. The detection limits of decorin in the plasma ranged between 31.3 and 2000 pg/mL. All experiments were repeated at least 3 times, and the mean decorin levels (ng/mL) were calculated and presented.