Bio plex manager 3

Heart tissue and serum levels of cytokines and chemokines were determined with a Bio-Plex Pro Mouse Cytokine Grp I Panel 23-plex (Luminex Corporation, Austin, TX, USA) using Bio-Plex 200 System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s guidelines. A total of the following 23 cytokines and chemokines: G-CSF, GM-CSF, Eotaxin, INF-g, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, KC, MCP-1, MIP-1a, MIP-1b, RANTES, and TNF-α were assayed with a single assay according to the kit instructions. Briefly, serum samples and heart tissue protein lysate samples were incubated with the microbeads for 30 min. After the incubation, the detection antibodies were introduced subsequently to the tests and incubated for 30 min. Further, the plates were washed with a wash buffer and the supernatant was removed and Streptavidin-Phycoerythrin was added to each well. Next, the plates were shaken at 850 rpm for 10 min and then washed and the supernatant was removed again. Finally, an assay buffer was added to each well to resuspend the pellet, and then the plate was read in a calibrated Bio-Plex machine. Concentrations of above cytokines and chemokines were analyzed with the Bio-Plex Manager 3.0 software (Bio-Rad, Hercules, CA, USA).