Uv vis microplate spectrophotometer

A Biotek microplate UV-Vis spectrophotometer equipped with UV KC junior software (Biotek) was used. The spectrum of the isolated protein fraction was measured at 200–790 nm using a quartz 96-well microplate. The results were expressed as absorbance units (AU).

Freshly collected leaves of the maize seedlings (Waza and Złota Karłowa cvs) were ground in liquid nitrogen. Total activity of the thioredoxin reductase (TrxR) was determined using the colorimetric Thioredoxin Reductase Assay Kit (Abcam, Cambridge, Great Britain; catalogue no. ab83463), following the manufacturer’s protocol. Portions of the powder (50 mg) were homogenized with 200 μL ice-cold Assay Buffer. Next, the samples were centrifuged at 10,000 × g for 15 min. (at 4 °C). The supernatant was collected and used for determination of TrxR activity. Measurement of TrxR activity was based on reduction of DTNB (5, 5′-dithiobis (2-nitrobenzoic) acid to TNB (5-thio-2-nitrobenzoic acid). The yellow color developed was measured at λ = 412 nm, using a microplate UV-Vis spectrophotometer (BioTek, Winooski, VT, USA). One unit (U) of TrxR activity was defined as the amount of enzyme that generates 1.0 μmol of TNB per minute at 25 °C. Total activity of TrxR in the maize samples was expressed as nmol of TNB per minute per milligram of protein. The protein content in the extracts was determined using Lowry method [54 (link)].

To determine the time course of irisin action, H9C2 cells (1000 cells/well) seeded in 96-well plates (Costar, 3595) were treated with hr-irisin, 50 nM of r-irisin, or control reagent for 0–4 days. In dose-response studies, the cells were cultured in the presence of 6 to 100 nM of r-irisin or control for 4 days. Cell proliferation at each time point and under different concentrations was then quantitated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay following the manufacturer’s protocol. Briefly, 10 μl of 5 mg/ml MTT solution (Sigma-Aldrich) was added to each well, and samples were incubated at 37°C. After 4 hours, the samples were centrifuged, and the absorbance of supernatants was measured at 570 nm using the UV/vis microplate spectrophotometer (BioTeK). The MTT assay was performed with 16 replicates in three independent experiments.