Bio plex human cytokine group assay kit

Cytokine measurement was performed simultaneously using the Bio-Plex Human Cytokine Group Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) in accordance with the manufacturer’s instructions. In brief, 50 μL of antibody-coupled beads per well were added to a flat-bottom plate and washed twice. Then, the samples (50 μL) were incubated with antibody-coupled beads for 30 min at room temperature. After washing three times to remove unbound material, the beads were incubated with 25 μL of biotinylated detection antibodies for 30 min at room temperature. Three washes were carried out to remove unbound biotinylated antibodies, and the beads were then incubated with 50 μL of streptavidin-PE for 10 min at room temperature. Following removal of excess streptavidin-PE by three wash cycles, the beads were re-suspended in 125 μL of assay buffer. The beads were then read on the Bio-Plex suspension array system, and the data were analyzed using Bio-Plex Manager software version 6.0 (Bio-Rad Laboratories).

Cytokine IL-17 (also known as IL-17A) and TNF-α measurements were performed simultaneously using the Bio-Plex Human Cytokine Group Assay kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. The principle of assay was followed: antibodies specific for human IL-17 or TNF-α coated on a 96-well plate; then, standards and samples were pipetted into the wells and IL-17 or TNF-α was bound to the wells by the immobilized antibody. The wells were washed and biotinylated, anti-human IL-17 or TNF-α antibodies were added. After flushing in triplicate for unbound biotinylated antibody, HRP-conjugated streptavidin was pipetted into the wells. The TMB substrate solution was added to the wells and color developed in proportion to the amount of IL-17 or TNF-α bound. The stop solution changes the color from blue to yellow, and the intensity of the color was measured at 450 nm by Bio-Plex suspension array system, with data analyzed using Bio-Plex Manager software version 6.0 (Bio-Rad Laboratories, Hercules, CA, USA).

The levels of the cytokines IL-4, IL-5, IL-6, IL-13, IL-17 and tumor necrosis factor alpha (TNF-α) were determined using the Bio-Plex Human Cytokine Group Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Antibodies specific for human IL-4, IL-5, IL-6, IL-13, IL-17 and TNF-α were immobilized or coated on 96-well plates, ensuring that the cytokines in the samples and standards were bound to these immobilized antibodies. After the cells were washed, they were flushed in triplicate for unbound biotinylated antibody, and horseradish peroxidase (HRP)-conjugated streptavidin was pipetted into the wells. 3,3’,5,5’-Tetramethylbenzidine (TMB) was added to the wells, and the color developed in proportion to the amount of cytokines. A stop solution was added to halt color development, and the intensity of the developed color (yellow) was measured at 450 nm using a Bio-Plex suspension array system. The data were analyzed using Bio-Plex Manager software version 6.0 (Bio-Rad Laboratories).