Powerwave xs spectrophotometer

The Cit-H3 concentrations were determined using an enzyme immunoassay using the ELISA method. Cayman's Citrullinated Histone H3 (Clone 11D3) (Cat. No. 501620, Cayman) ELISA kit was used in the investigation. Serum served as the research's initial stage. The test was performed in doubles. The final measurement was placed at a wavelength of λ = 450 nm on a BioTek Power Wave XS spectrophotometer by Bio-Tek Instruments. Calculation of the Cit-H3 concentration in the test tubes was performed with the KCjuniorWin computer program, using the standard curve method.

Plasma CK reflects the leak of CK from muscles into the blood and is a classic systemic measure of damage and necrosis of dystrophic muscles [46 (link)]. CK levels were measured using a CK-NAC kit (Randox Laboratories, Crumlin, United Kingdom) and analysed kinetically using a BioTek Powerwave XS Spectrophotometer (Currumbin, QLD, Australia) using the KC4 (V34) program.

Oil Red O staining was used to determine intracellular lipid content in cells. Cultures of adipocytes and hepatocytes were washed with PBS and fixed with paraformaldehyde 7% (Sigma-Aldrich) for 1 hour. Then, cells were dipped in isopropanol 60% before being completely dried and stained with filtered 5% Oil Red O in isopropanol for 10 minutes at room temperature; they were then washed up to 4 times with distilled water. Pictures were taken using an inverted microscope Leica DM IL LED. After they were completely dried, cells were dipped in 100% isopropanol and incubated up to 10 minutes to elute Oil Red O. Optical density was measured at 500 nm using a PowerWave XS spectrophotometer (BioTek Instruments).

Protein quantification was determined by using a commercial Micro BCA Protein Assay Kit (Pierce, Waltham, MA, USA, 23235). Protein extracts were resuspended in 2% SDS and incubated overnight at 37 °C to completely dissolve the protein pellet. 1:100 dilutions were produced and incubated with reagent solution at 37 °C for 2 h. After that, absorbance was measured at 625 nm in a PowerWave XS spectrophotometer (Bio-Tek, La Puenta, CA, USA). A standard curve with increasing concentrations of BSA was prepared and used for quantification.

Total RNA was obtained from 0.1 g from gastrocnemius muscle using TriPure Isolation Reagent (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s instructions. Total RNA was quantified using the Take3 Microplate in a PowerwaveXS spectrophotometer (BioTek, Winooski, VT, USA). A 1-μg sample of total RNA was reverse transcribed to cDNA using 25 U MuLV reverse transcriptase in a 5-μL retrotranscription mixture (10 mM Tris–HCl pH 9.0, 50 mM KCl, 0.1, 2.5 mM MgCl2, 2.5 μM random hexamers, 10 U RNase inhibitor, and 500 μM of each dNTP) for 60 min at 42 °C in a Gene Amp 9700 thermal cycler (Applied Biosystems, Alcobendas, Madrid, Spain). cDNA solutions were diluted 1/10, and aliquots were frozen (−20 °C) until analyzed. Real-time PCR was carried out using SYBR Green technology in a LightCycler rapid thermal cycler (Roche Diagnostics, Mannheim, Germany). The amplification program consisted of a preincubation step for denaturation of template cDNA (95 °C, 10 min) followed by 45 cycles consisting of a denaturation, an annealing, and an extension step under the conditions given in Table 1. After each cycle, fluorescence was measured at 72 °C.

BMMSCs were inoculated into 96-well plates (2 × 10³ per well). After cell culture to the target time, 10 μl of CCK-8 solution was added to each well for 1 h and incubated at 37°C with 5% CO₂. Absorbance was measured at 450 nm using a PowerWave XS spectrophotometer (BioTek, Winooski, VT, USA).

A MTT assay was performed to ensure that any observed changes in the glucose utilization or uptake were not due to changes in the cell viability. The MTT assay is based on the reduction of a yellow water-soluble tetrazolium salt to an insoluble purple formazan product. The purple-colored formazan crystals are dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 540 nm. The concentration of formazan is then related to the number of healthy viable cells [110 (link),111 (link)]. Briefly, the remaining treatment medium from the assay described above was aspirated from all wells and 100 μL of the complete medium, containing 0.5 mg/mL MTT, was added. Thereafter, the cells were incubated for 1 h at 37 °C and a MTT medium was removed and 100 μL DMSO was added to each well. The absorbance was read at 540 nm using a BioTek^® PowerWave XS spectrophotometer (Winooski, VT, USA).