Sandwich elisa

Cell or nuclear extracts were prepared from both treated and control HepG2 cells in cell lysis buffer and from ARSB-null and control mouse liver. GPNMB was detected in the whole cell lysate, whereas MITF concentration was determined in the nuclear extract, prepared as above. Nuclear MITF (MyBioSource, Inc., San Diego, CA) and cellular GPNMB (R&D Systems, Minneapolis, MN) were measured in cell and tissue samples using sandwich ELISAs (R&D). Microtiter plates were coated with a capture antibody to the molecule of interest. Samples and standards were added to the wells of the microtiter plate, and the antigen in the lysates was captured by the coated antibody on the plate and detected with biotinylated antibody and HRP-conjugated streptavidin. Hydrogen peroxide / tetramethylbenzidine (TMB) substrate was used to develop the color which was proportional to the bound HRP. The reaction was stopped and the optical density of the color was read at 450 nm in a plate reader (FLUOstar, BMG Labtech, Cary, NC). The concentration in the sample was extrapolated from a standard curve derived using known concentrations.

The inflammatory state of the mice exposed to Pa-LPS and/or SAHA was quantified by measuring BALF or serum cytokine levels (IL-6) and neutrophil (MPO, myeloperoxidase) activity by sandwich ELISAs (R&D and Hycult Biotechnology respectively), as recently described [10 (link), 16 (link)]. The flow cytometry analysis of BALF samples from each murine experimental group was performed as previously described [10 (link), 16 (link), 21 (link)] and was used to quantify the percentage changes in number of CD4 + FoxP3+ T cells in BALF-cells. The BD FACS Caliber instrument and BD Cell Quest Pro software was used for acquisition and analysis of the data.

Cytokine analyses on supernatants were completed with a multiplex bead-based assay (Bio-Rad Laboratories, Hercules, CA, USA) these data were confirmed via sandwich ELISAs (R&D Systems, Minneapolis, MN, USA). Whole cell lysates were generated using M-PER digestion buffer (Thermo Fisher Scientific Inc, Waltham, MA, USA) and were analyzed for cytokine production using identical ELISAs. Protein phosphorylation was measured using a cell-based colorimetric ELISA system which compared phosphorylated (Ser32) to total levels of I-κBα, phosphorylated (Ser73) to total levels of c-Jun and dual phosphorylated to total p38MAPK (SABiosciences, Frederick, MD, USA).

Cytokine concentrations were estimated in culture supernatants using the Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit (BD) or sandwich ELISAs (R&D), as indicated.