Macrosep centrifugal devices

hFOB cells were cultured in a 100-mm culture dish. Culture medium was replaced by FBS and phenol red-free medium. 3-MC (10 μM) or β-NF (10 μM) was added, and after 24 h, conditioned medium (total 30 mL) was collected. AhR antagonist (10 μM CH-223191) and 3-MC were simultaneously added to the cell culture medium. Conditioned medium was concentrated to a 5 mL volume using Macrosep Centrifugal Devices (Pall Corporation, Port Washington, NY, USA). In this study, we employed the Human Cytokine Antibody Array 5 (RayBiotech, Inc., Norcross, GA, USA) in order to identify the cytokines released from hFOB cells with/without AhR ligand treatment. The membranes were incubated with biotin-conjugated anti-cytokines (provided with the kit) and developed with horseradish peroxidase-streptavidin and chemiluminescence. Protein dots were visualized with a Las-1000 cooled CCD-camera chemiluminescent image analyzer (Fuji Photo Film, Tokyo, Japan).

Protein fractions with highest purity were pooled and concentrated using Macrosep Centrifugal Devices (Pall Corporation, USA) with 10 kDa cut-off. Concentrated protein was dialysed against 50 mM sodium phosphate pH 8.0. The purified proteins were quantified using Pierce BCA protein assay kit (Thermo Scientific, USA). Slope from the standard calibration curve of bovine serum albumin (BSA) was used for quantification of protein. Dot blot assay was performed to detect the activity of purified SpChiD fusion chimeras as described by Purushotham et al. [23 (link)]. The lytic zones were visualized as dark blue spots in the gels under UV transilluminator.