Biorad bioplex hts

The protein levels of various pro-inflammatory cytokines/chemokines; RANTES, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), IL1-β, and TNF-α were measured using multiplex cytokine assay kit (Bio-Rad, CA, USA) as per the manufacturer’s protocol. Briefly, plasma samples were centrifuged at 3000 g for 10 min and the supernatants were diluted with three volumes of sample diluent. Next, 50 μl of each sample and standards were mixed with magnetic beads and incubated on a shaker at room temperature for 30 min. The beads were washed and 25 μl of detection antibody was added to each well followed by incubation for 30 min at room temperature. Furthermore, the samples were washed and incubated with 50 μl streptavidin-PE conjugate for 10 min. Finally, 125 μl of the assay buffer was added and the samples were analyzed using Biorad Bioplex HTS (Bio-Rad, CA, USA). The concentrations of the cytokines were determined with Bio-plex manager 5 using 5-PL statistics using standard curve.

Plasma levels of various pro-inflammatory cytokines/chemokines including RANTES, IL-6, IL-8, MCP-1, and TNF-α were measured as described previously [19 (link)] using the multiplex cytokine assay kit (Bio-Rad, CA, USA). Briefly, plasma samples were centrifuged and the supernatants were diluted with sample diluent (1:3). Next, 50 µl of each sample was used to determine the levels of these cytokines/chemokines using Biorad Bioplex HTS (Bio-Rad, CA, USA). The levels of cytokines were determined using the standard curve.