Human cytokine array panel a membranes

HSC3 cells were plated on to a 10-cm cell culture plate at a density of 1 × 10⁶ cells on the day before the experiment. The cells were treated with or without FW for 30 s. After washing, the cells were cultured for another 18 h. The culture supernatants were collected and subjected to cytokine array (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instruction. Briefly, the culture supernatants were centrifuged at 10,000 × g for 2 min and the supernatants were transferred to new tubes. Human cytokine array panel A membranes (R&D Systems) were incubated with FW-treated (Sup+) or -untreated (Sup-) HSC3 cell culture supernatants. After washing, the membranes were further incubated with a detection Ab cocktail for 1 h. Spot detection was performed with streptavidin-horseradish peroxidase incubation, followed by the use of the Enhanced Chemiluminescent (ECL) kit (GE Healthcare, Tokyo, Japan). The intensity of the spot was measured by the NIH Image software. The Sup+ and Sup- were subjected to IL-1α concentration measurements using the DuoSet enzyme-linked immunosorbent assay (ELISA) development system (R&D Systems). The absorbance was measured on a microplate reader (model 3550; Bio-Rad, Tokyo, Japan).

Neutrophils were pre-incubated in the presence or absence of GSK484 and stimulated with mycobacteria at a MOI of 1 for 4 h at 37 °C. The culture supernatants were collected and subjected to cytokine array analysis (ARY005B, R&D Systems, Minneapolis, MN, USA) according to manufacturer's instructions. Briefly, human cytokine array panel A membranes (R&D Systems) were incubated with cell culture supernatants. After washing, the membranes were further incubated with a detection Ab cocktail for 1 h. Spot detection was performed with streptavidin–horseradish peroxidase. The intensity of the spot was measured with ImageJ software (U.S. National Institutes of Health; http://rsb.info.nih.gov/ij/).