Human ccl18 parc duoset elisa kit

The supernatants of macrophages treated with or without rISG15 were incubated with detection membranes at 4°C overnight. The procedure was performed according to the manufacturer’s protocol for the RayBio Human Cytokine Antibody Array (Raybiotech, catalog no. AAH-CYT-1000). The protein levels of M1- and M2-polarizing cytokine were detected by using Human TH1/TH2/TH17 Cytokine CBA Kit (BD Bioscience, Catalog no. 560484), which contained a cytokine panel of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ. The protein level of CCL18 was measured by using Human CCL18/PARC DuoSet ELISA Kit (R&D, Catalog no. DY394-05). The procedures were performed according to the manufacturer’s protocols.

TNF-α, IL-6, IL-8, and IL-1β production was assessed by means of the FlowCytomix bead-based multiplex system (eBioscience) as described before^{52 (link)–54 (link)}. Briefly, 10 μl of sample or standard was mixed with 10 μl of a cocktail of beads coated with primary antibodies for the detection of the cytokines of interest and 20 μl of a cocktail of biotin-conjugated secondary antibodies in a FACS tube and incubated for 2 h at room temperature in the dark. Tubes were then washed twice with 400 μl each of assay buffer (provided in the kit) to removed unbound beads and antibodies. 20 μl of diluted streptavidin-phycoerythrin (PE) conjugate was then added to each tube and the samples/standards incubated for 1 h at room temperature in the dark. Samples were washed twice with assay buffer as before and resuspended in 400 μl assay buffer, stored at 4 °C, and analysed on a Beckman Coulter FC500 flow cytometer within 24 h. Results were analysed using the eBioscience FlowCytomix Pro 3.0 software.
CCL18 production was measured using the Human CCL18/PARC DuoSet ELISA kit (R&D Systems) as per the manufacturer’s instructions.
IL-1RA and IL-10 production were measured using the ProcartaPlex bead-based luminex system (Affymetrix eBioscience) as per the manufacturer’s instructions. Plates were read on a Bio-Rad Bio-Plex 200 system and results analysed using the ProcartaPlex Analyst 1.0 software.