Elisa kit

To examine cytokine (TNF-α and IL-6) secretion, we collected the supernatant of the non- infected, infected, and phloretin-treated groups, and then TNF-α and IL-6 were examined using ELISA kits (Invivogen, USA) according to the manufacturer’s instructions.

The supernatants were collected from primary peritoneal macrophages or peripheral blood in the indicated experiments. The concentrations of IFN-α, IFN-β or TNF-α cytokine were detected with ELISA kits according to the manufacturer’s protocols (InvivoGen). The capture antibody was added to the plate, and incubated overnight at 4°C. The plate was washed three times using wash buffer, then added blocking buffer to each well and covered at room temp for 1 h. The samples or standards were added to the plate and incubated at room temp for 1 h after removing the blocking buffer. The plate was washed 3 times 5 min each, then added biotinylated detection antibody and incubated at room temp for 1 h. The enzyme conjugate was added to each well and incubated at room temp for 1 h after washing three times 5 min each. The plate was washed three times 5 min each, added substrate solution to each well and incubated at room temp for 30 min. The absorbance was measured by appropriate hardware and analyzed the data.

Enzyme-linked immunosorbent assay (ELISA) kits (InvivoGen) were used to measure IFN-α and IFN-β in the plasma following the manufacturer’s instructions. The plates were scanned with FLUOstar Omega microplate reader. Limits of detection: IFN-α, 7.8–500 pg/ml and IFN-β, 15.6–1000 pg/ml. A Q-plex array (Quansys Biosciences) was used to determine cytokine and chemokine levels in plasma. The plate was scanned with Odyssey Infrared Imaging System (LI-COR Biosciences) and analyzed with Q-View (Quansys Biosciences). Limits of detection: TNF, 4.12–3000 pg/ml; IL-1β, 19.2–14000 pg/ml; IL-5, 6.86–5000 pg/ml; IL-6, 6.86–5000 pg/ml; IFN-γ, 10.97–8000 pg/ml; CCL1, 5.6–4000 pg/ml; CCL2, 4.12–3000 pg/ml; CCL22, 5.6–4000 pg/ml.