P nitrophenyl phosphate disodium salt hexahydrate

Nitrite levels in infected cell culture supernatants were measured using the Griess reagent assay. Briefly, cell supernatants were incubated with 1% sulfanilamide in 2.5% phosphoric acid for 10 minutes at room temperature in the dark, followed by 0.1% naphthyl-ethylene-diamine in 2.5% phosphoric acid for another 10 minutes. Cytokines and chemokines from BMDM cell culture and organ homogenates were examined using the standard sandwich enzyme-linked immunosorbent assay (ELISA) protocol. Capture and biotin antibodies were obtained from either BD Biosciences (Franklin Lakes, New Jersey), BioLegend (San Diego, California), or R&D Systems (Minneapolis, Minnesota) using either KPL TMB Microwell Peroxidase Substrate (SeraCare Life Sciences, Milford, Massachusetts) for streptavidin-HRP conjugates or 1 mg/ml p-nitrophenyl phosphate disodium salt hexahydrate (Sigma Aldrich, St. Louis, Missouri) for streptavidin-AP conjugates. Optical density was measured using the VersaMax™ microplate spectrophotometer (Molecular Devices, San Jose, California).

Hank’s Balanced Salt Solution (HBSS), HEPES, Tween 20, phosphate buffered saline (PBS) tablets, acetone for HPLC > 99.9% and p-nitrophenyl phosphate disodium salt hexahydrate (PnPP) tablets were purchased from Sigma-Aldrich, St. Louis, MO. N-(3-maleimidylpropionyl) biocytin (MPB) was from Invitrogen, Carlsbad, CA and Immobilizer Streptavidin C8 and MaxiSorp 96-well plates were purchased from Nunc, Roskilde, Denmark. Polyclonal rabbit anti-human angiotensinogen antibody (pAb) (catalogue number LS-C80597) was purchased from Lifespan Bioscience (Seattle, WA). Polyclonal goat anti-mouse IgG (Fab specific) alkaline phosphatase (AP) antibody (catalogue number A1293), polyclonal goat anti-rabbit IgG (whole molecule) AP (catalogue number A9919), bovine serum albumin (BSA) and angiotensinogen from human plasma were purchased from Sigma-Aldrich. Mouse anti-human angiotensinogen monoclonal antibody (clone 369439) was purchased from R&D Systems, Minneapolis, MN.

Serum cytokines (IL-4 and TNF-α) and total IgE titer were measured as follows. Blood was collected in serum separator tubes (BD Biosciences) and centrifuged at 8,000 g for 10 min at 4°C to separate serum. The Nunc MicroWell 96-Well Microplates (Thermo Fisher Scientific) were coated with 0.5 ng/ml capture antibody in PBS and incubated overnight at 4°C. Plates were washed and blocked with 2% (w/v) milk powder for 2 hr at 37°C, and samples were loaded and incubated overnight at 4°C. Detecting, biotin-labeled antibodies were added and incubated for 2 hr at 37°C, and then, avidin-horseradish peroxidase or avidin-alkaline phosphatase was added and incubated for 1 hr at 37°C. Plates were developed by adding TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, US) or p-nitrophenyl phosphate disodium salt hexahydrate (Sigma), respectively. The absorbance was read at 450 or 405 nm using a VersaMax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, US).