Varioskan lux multimode microplate reader

In parallel with cfDNA, IL-6, and PGF2α levels were measured to confirm the inflammatory response induced by exercise. IL-6 was measured using Human IL-6 Quantikine HS ELISA (Cat.HS600C; R&D Systems, Minneapolis, MN, USA). The assay was performed according to the product protocol, and 100 μl of serum sample was used without dilution. The absorbance was measured at 450 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). A 4PL curve was generated from the absorbance of the standard solution using ImageJ, and the level of IL-6 in each serum sample was calculated. PGF2α was measured using a Prostaglandin F2 alpha ELISA Kit (Cat.KA0310; Abnova, Taipei, Taiwan). The manufacturer's instructions were followed, and 100 μl of a 10-fold diluted serum sample was used. The absorbance was measured at 405 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). The binding rates of the standard solution and each serum sample were calculated, and a 4PL curve was generated from the binding rates of the standard solution using ImageJ. The PGF2α concentration in each serum sample was calculated.

For ROS detection, confluent HUVEC were cultured in a 96-well black plate (Greiner Bio-One, Kremsmunster, Austria) and, at the end of the experiments, they were incubated for 30 min with 10 mM 2′-7′-dichlorofluorescein diacetate (DCFH) solution (Sigma-Aldrich). The DCFH dye emission was monitored at 535 nm (excitation λ = 484 nm) using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). Then, the cells were fixed in Phosphate Buffered Saline (PBS) containing 3% paraformaldehyde (PFA) and 2% sucrose (pH 7.6) for 30 min and, after extensive washing, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI), which was used to stain the nuclei (1:10,000). DAPI florescence (λ_ex/em = 350/470 nm) was monitored using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific) and was used to normalize the DCFH dye emission [7 (link)]. The results are the mean of three independent experiments performed in triplicate ± SD.

The TAS levels in the blood and tissue samples were measured using a commercial kit (Rel Assay Diagnostics, Turkey) with a spectrophotometric method (Thermo Scientific, Varioskan™ LUX multimode microplate reader, USA). Briefly, the antioxidants in the samples reduced the dark blue green 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical to the colorless reduced form of ABTS. The change in absorbance at 660 nm was related to the total antioxidant level of the sample. Total antioxidant activities were reported as mmol Trolox equiv/L of the samples.
The TOS levels in the samples were measured using a commercial kit (Assay Rel Diagnostics, Turkey) and spectrophotometer (Thermo Scientific, Varioskan™ LUX multimode microplate reader, USA). Briefly, oxidants present in the samples oxidized the ferrous ion chelator complex to iron ions. While the oxidation reaction was prolonged by enhancer molecules abundant in the reaction medium, the ferric ion formed a color complex with chromogen in an acidic environment. The intensity of color formed was related to the total amount of oxidant molecules present in the samples. The results are reported as μm H₂O₂ equivalent/L.