Gallios 3 l 10c

Cytokine levels were determined in supernatants collected from the PBMC proliferation assays (anti-CD3 + anti-CD28 stimulated cells) after 6 days. All cytokine measurements were carried out with the human Th1/Th2/Th9/Th17/Th22 13plex bead array Kit (eBioscience, BMS817FF) according to the manufacturer's instructions. Briefly, samples were incubated with a bead mixture and biotin-conjugated cytokine specific antibody mixture light protected for 2 hours at room temperature. Samples were washed twice by adding assay buffer and spinning down with 200 g for 5 min. Staining with Streptavidin-PE solution was carried out under light protected conditions for 1 hour at room temperature, followed by 2 washing steps. Cytokines were assessed by flow cytometry (Gallios 3 L 10C, Beckman Coulter) and analyzed with FlowCytomixPro software (eBioscience).

ROS activity was measured with the ROS-ID Total ROS detection kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s protocol. Briefly, cells were cultured for 4 days and collected. Subsequently, cells were washed and resuspended in ROS detection solution. Samples were incubated in the dark for 30 min and analyzed by flow cytometry at FL1 (490/525 nm) using a cytometer (Gallios, 3L10C, BECKMAN COULTER, Tokyo, Japan). The data were evaluated by triplicate measurements.