Synergy ht 96 well plate reader

For the two-hybrid system, E. coli cells were grown at 37 °C with aeration in MOPS media, in the presence of either 10 or 100 μM Fe(III). Gene expression of the fusion proteins in the reporter strain was induced by the addition of 5 μM IPTG, at an OD₆₀₀ = 0.25. Culture samples were taken after 4 h, permeabilized with 0.15 % sodium dodecyl sulfate (SDS) and 1.5 % chloroform in Z-buffer (Miller, 1972 ).
β-galactosidase activity was assayed by following the catalytic hydrolysis of the chlorophenol red-β-D-galactopyranoside (CRPG) substrate (Sigma-Aldrich). The change in absorbance at 570 nm was read continuously using a Synergy HT 96-well plate reader (BioTek, Winooski, VT). β-galactosidase activity, expressed as arbitrary units (AU), was calculated using the slope of absorbance curve normalized with the initial cell density. The assays were performed in triplicate.

IL-1β, IL-6, IL-7, IL-8 (CXCL8), IL-10, IL-12p70, tumor necrosis factor alpha (TNFα), CXCL1 (growth-related oncogene), CXCL10 (human interferon-inducible protein 10), CCL2 (monocyte chemoattractant protein-1), CCL3 (macrophage inflammatory protein-1α), CCL4 (macrophage inflammatory protein-1β), and CCL22 (macrophage-derived chemokine) were quantified with bead-based multiplex assays (HSCYTO-60SK and MPXHCYTO-60K, Millipore, Billerica, MA). IL-33 (R&D Systems, Minneapolis, MN), transforming growth factor beta-1 (TGF-β1) (Promega, Madison, WI), neutrophil elastase (Abcam, Cambridge, MA), myeloperoxidase (R&D Systems) and lactoferrin (Abcam) ELISA were performed according to manufacturer instructions. Concentrations were determined with a BioTek (Winooski, VT) Synergy HT 96-well plate reader using manufacture recommended wavelengths and standard curve fit.