Il 8 elisa kit

NCI-H292 cells, a human pulmonary muco-epidermoid carcinoma line, were acquired from the American Type Culture Collection (CRL-1848; ATCC, Manassas, VA, United States). Early passages (passage number 7–20) were used for all experiments. Cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640; Hyclone, GE Healthcare, United Kingdom) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100 units/mL penicillin plus 100 μg/mL streptomycin (Hyclone) at 37°C under a humidified 5% CO₂ atmosphere. For enzyme linked immunosorbent assay (ELISA) of IL-6 production, NCI-H292 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells for 16 h. They were then transferred to reduced-serum medium (0.1% FBS). After a 16 h incubation period, the cells were treated with different fractions from Xinyi or the seven purified lignans from the CHCl₃ extract for 2 h before adding CSC (20 μg/mL). After addition of CSC, the cells were further incubated for 12 h. The presence of cytokines in the supernatant was analyzed using IL-6, IL-1β, and IL-8 ELISA kits according to the manufacturer’s instructions (R&D, Minneapolis, MN, United States).

Media from NHEKs stimulated with UVB radiation were harvested and centrifuged for 15 min at 4 °C. The supernatants were freeze-dried and used for multiple cytokine measurements with a human cytokine array C3 (Raybiotech, GA, USA), according to the manufacturer’s instructions. The IL-6 and IL-8 levels were quantified using IL-6 and IL-8 ELISA kits, respectively, according to the manufacturer’s instructions (R&D Systems).

The concentrations of MIF, HPA-1, CD138, MMP-9, IL-6 and IL-8 in the serum or cell culture medium were measured using human MIF, HPA-1, CD138, MMP-9, IL-6 and IL-8 ELISA kits (R&D Systems) following the manufacturer’s instructions. The concentrations of MIF, HPA-1, MMP-9 and CD138 in the serum or peritoneal lavage fluid of mice were measured using mouse MIF, HPA-1, MMP-9 and CD138 ELISA kits (BlueGene Biotech, Shanghai, China). NS1 ELISA was carried out using paired anti-NS1 antibodies prepared in our laboratory and was quantified by the addition of 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (R&D Systems).

The medium was harvested from NHEKs, stimulated with UVB radiation, and centrifuged for 15 min at 4 °C. The supernatants were freeze-dried and used for multiple cytokine measurements with Human Cytokine Array C3 and Human Cytokine Array C4 Kits (RayBiotech, Norcross, GA, USA) according to the manufacturer’s instructions. The arrays were imaged with a Fujifilm LAS-4000 imager. Image analysis was performed using LI-COR Image Studio Lite. Each dot was assigned a value, and the average of two measurements was calculated for each array. The average density values for each cytokine in each condition were then averaged for each array to calculate the fold change. IL-6, IL-7, and IL-8 levels were quantified using IL-6, IL-7, and IL-8 ELISA kits, respectively, according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

The concentration of different cytokines was analyzed by commercial ELISA kits according to the manufacturer’s instructions. The IL-1β, IL-1ra, IL-6, TNFα, IL-10 and IL-8 ELISA kits were obtained from R&D Systems (Abingdon, United Kingdom). For the pro-IL1β measurement, macrophages were lysed by repeated freeze thaw cycles and then the supernatant was collected [39]. The experiments were performed with both PBMCs and macrophages derived from peripheral blood mononuclear cells (PBMC-DMs) were used from at least five independent donors.