Sulfanilic acid

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA and PBS were obtained from GIBCO, Invitrogen, USA. Nitro blue tetrazolium (NBT) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) were obtained from SERVA, Germany. Sodium nitrite, phosphoric acid, sulfanilamide, sulfanilic acid, sulfosalicylic acid, and 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich, USA. Petroleum ether (boiling point ranges: 40 °C-60 °C), methylene chloride, ethyl acetate, chloroform, acetic acid, dimethyl sulfoxide (DMSO), HPLC grade methanol, highly polymerized calf thymus DNA (CT-DNA), human serum albumin (HSA, lyophilized powder, free fatty acid r0.007 %, purity r96 %, Catalogue No. A1887), phosphate buffered saline (PBS) tablets, ethidium bromide, fetal bovine serum (FBS) and 7-Aminoactinomycin D (7-AAD) were purchased from Sigma-Aldrich, (Steinheim, Germany). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Brea, CA, USA).

The activated coal (SX+) was obtained from NORIT. The reduced graphene oxide N002-PDR (RGO) was obtained from Angstron Materials. The carbon multi-walled nanotubes (MWCNT) were obtained from Nanocyl (Belgium) (NC 7000 Thin MWCNTs, 95+% C purity). The carbon black (CB) was obtained as 250 G type from IMERYS GRAPHITE & CARBON. The carbon nanofibers (CNF; LHT type) were obtained from Applied Sciences Inc (USA). The graphene nanoplatelets (GNP; M15, and C750 types) were purchased from Sigma-Aldrich. Sulfanilic acid (99%), isopentyl nitrite (96%), D-(+)-cellobiose (≥99%), cellulose (reference C6288, crystalline and high purity) were also supplied by Sigma-Aldrich and used as received.

The colorimetric Griess assay was used to measure nitrite (Lam et al., 2017 (link); Lively and Schlichter, 2018 (link)). Briefly, microglia (8 × 10⁴ cells/coverslip; 17–19 independent cultures for each sex at P1; 9–13 independent cultures for each sex at P21) were incubated for 24 h with I+T or IL-4 (as above) and then aliquots of the supernatants were added to 96-well plates containing 1% sulfanilic acid (Sigma-Aldrich; Cat#86090). After adding 0.1% N-(1-naphthyl)ethylene diamine dihydrochloride (Sigma-Aldrich; Cat#222488), the plates were incubated for 30 min in the dark. The resulting color change (absorbance at 570 nm) was quantified using a plate counter (Victor³ 1420, Perkin Elmer, Woodbridge, ON, Canada), and the nitrite concentration calculated by interpolation from a standard curve.

Folin-ciocalteau (FC reagent), gallic acid, 2,2-azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) diammonium salt (ABTS), sodium nitrite, sulfanilic acid, acetic acid, sodium citrate, ascorbic acid and ferrous sulfate heptahydrate (FeSO₄) were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). D-Catechin was purchased from MP Biomedicals, LLC (Illkich, France). Hydrogen peroxide 30% was obtained from Daejung Chemicals and Metals Co. (Gyeonggi-do, Korea). pBR322 DNA and 6× DNA loading dye were purchased from Thermo Fisher Scientific Inc. (NYSE: TMO, Waltham, MA USA; Gyeonggi-do, Korea). RAW 264.7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). DMEM, penicillin-streptomycin and fetal bovine serum (FBS) were purchased from GIBCO BRL (Life Technologies, Grand Island, NY, USA). All chemicals were used without any further purification.

Nitrite-scavenging activity was evaluated based on the absorbance at 520 nm using a UV-spectrophotometer according to the method reported by Kato et al. (1987 ). One milliliter of 1 mmol/L NaNO₂ (Sigma Co.) solution was added to 1 mL of each sample, and the resulting mixtures were adjusted to pH 2.5 using 0.1 N HCl and 0.2 N citric acid solutions. Each sample was allowed to react at 37°C for 1 h, after which 1 mL of each sample was taken from the solution and mixed thoroughly with 3 mL of 2% acetic acid and 0.4 mL of the Griess reagent. The solutions were stored at room temperature for 15 min. The Griess reagent was prepared by mixing an equal amount of 1% sulfanilic acid (Sigma Co.) and 1% naphthylamine (Sigma Co.), which were made with 3% acetic acid. Nitrite-scavenging activity was calculated according to equation (1).

Escherichia coli LPS (O128:B12, Sigma-Aldrich, Castle Hill, Australia), sulfanilic acid, N-(1-Naphthyl) ethylenediamine (NED), 85% orthophosphoric acid sodium nitrite (NaNO₂), and HPLC grade solvents were obtained from Sigma Aldrich (St. Louis, MO, USA). The mouse TNFα ELISA kit was purchased from BD biosciences (Sparks, MD, USA). Penicillin–streptomycin solution, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, and l-glutamine were from Life Technology Australia (Mulgrave, VIC, Australia). The two cell lines, RAW264.7 mouse macrophages and 3T3 Swiss albino (ATCC^® CCL92™) cell lines, were purchased from the American Type Culture Collection (ATCC^®, Manassas, VA, USA).