Spectra mr

At the end of the incubation time, cell supernatants were collected, centrifuged, and stored at −70 °C until tested. IL-10 and TGF-β1 levels in CD4⁺CD25⁺ Treg culture supernatants were measured using ELISA kits, according to the manufacturer’s instructions. To determine T-cell cytokine production, culture supernatants from CD4⁺CD25⁺ Tregs/CD4⁺CD25⁻ T-cell cocultures were collected, and IL-2, IL-4, as well as IFN-γ levels were measured with ELISA. Microplate reader (Spectra MR, Dynex, Richfield, MN) was used to obtain the results.

Cell viability was determined using the Cell Titer 96^®Aqueous One Solution Reagent (MTS assay, PROMEGA, United Kingdom). Cells were cultured in a 96-well plate. Before measuring, each well medium was replaced by 100 μl of new medium and we added 20 μl of reagent, then we cultured the cells for 3 h at 37°C, 5% CO₂. A blank well with the same volume of medium but without cells was as a control well. The optical density (OD) was measured by microplate spectrophotometer (Spectra MR, Dynex, United States).

To examine the proliferation of PE-DBMSCs the cells were seeded at a density of 5 × 10³ per well in 96-well tissue culture plates containing complete DBMSC culture medium and incubated for 24 h at 37 °C in a cell culture incubator. Cell proliferation was evaluated using a MTS kit (#G5421, CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay, Promega, Germany) according to the manufacturer’s instructions. Briefly, MTS solution was added into each well of the 96 well assay plate containing DBMSCs in complete culture medium with or without H₂O₂, and incubated at 37 °C in a cell culture incubator. After 4 h, the absorbance at 490 nm was recorded using an ELISA plate reader (Spectra MR, Dynex Technologies, Denkendorf, Germany), and results were presented as means (±SD) obtained from triplicate samples. MTS solution in a medium not exposed to cells was also used as blank. Experiments were performed with triplicate samples and repeated five times using cells from passages 3–5 of five independent preparations of PE-DBMSCs.