N 1 napthylethylenediamine dihydrochloride

Nitric oxide production was assessed by using the Greiss Assay (Promega, Madison, WI, USA) following the manufacturer's protocol. Detection of nitrite was performed in 96-well plates, BV2 cells and PM (5 × 10⁵ cells/well) were incubated in DMEM containing 2% FBS alone or in combination with SC-CM, and BMSCs-CM, and SCI-CM, and SCI-CM + BMSCs-CM, ratio 2:1/DMEM:CM for 24 hours. NO was detected in the 50 μL of culture supernatant from each sample in triplicate and added with the same volume of Griess reagent (1% sulfanilamide/0.1% N-1- napthylethylenediaminedihydrochloride/2.5% phosphoric acid; all from Sigma-Aldrich, St. Louis, MO, USA). Absorbance was read at 530 nm (MRX II microplate reader, Dynex Technologies, VA, and USA) after 15 minute incubation. Nitrite concentration was calculated with reference to a standard curve of freshly prepared sodium nitrite (0 to 100 μM). All treatments were completed at least three times and data were expressed as mean μM concentration of NO₂ ± SEM.

Ethanol was purchased from Chem-Supply (Gillman, SA, Australia); bovine serum albumin, lipopolysaccharide (E. coli serotype-0127:B8), EDTA, N-(1-napthyl) ethylenediamine dihydrochloride, benzylpenicillin G sodium salt, resazurin sodium salt (10%), streptomycin, sulphanilamide, 3,3′,5,5′-tetramethylbenzidine (TMB), trypan blue, and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). GIBCO, fetal bovine serum (FBS), and glutamine were purchased from Life Technologies (Mulgrave, VIC, Australia). Murine interferon-γ (IFN-γ) and TNF-α ELISA kits were purchased from PeproTech Asia (Rehovot, Israel). Citric acid and monosodium dihydrogen carbonate (NaH₂CO₃) were from AJAX Chemicals (Auburn, NSW, Australia). Tween-20 was from Amresco (Solon, Ohio, USA). MEthanol, monosodium phosphate (NaH₂PO₄), disodium phosphate (Na₂HPO₄), sodium chloride (NaCl), and sulfuric acid (H₂SO₄) were from Merck (Darmstadt, Germany). Sodium carbonate (Na₂CO₃) was BDH brand supplied by Merck Pty. Ltd. (Kilsyth, VIC, Australia).

NO released from cells was converted to nitrite in the culture medium and was determined using Griess reagent [42 (link)]. Cells were cultured in phenol red free DMEM. After treatment with LPS for 16 h, aliquots (50 μL) of culture medium were transferred to 96-well plates and incubated with 50 μL of reagent A [1% (w/v) sulfanilamide (Sigma-Aldrich, St. Louis, MO) in 5% phosphoric acid] for 10 min at room temperature in the dark, and followed by incubation with 50 μL of reagent B [0.1%, w/v, N-1-napthylethylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO)] for 10 min at room temperature in the dark. The absorbance of samples was measured at a wavelength of 565 nm using the Synergy4 Plate Reader. Sodium nitrite (0–100 μM), diluted in culture media, was used to prepare the nitrite standard curve.

NO released from cells was converted to nitrite in the culture medium and determined using the Griess reagent protocol [25] (link). In brief, cells in 96-well plate were serum-starved in phenol red-free DMEM for 3 h, followed by incubation with specified concentrations of SF-E or inhibitors for 1 h, and then treated with IFNγ and/or LPS at 37°C for 16 h. Aliquots of the media (50 µL) were incubated with 50 µL of the reagent A (1% (w/v) sulfanilamide in 5% phosphoric acid, Sigma-Aldrich) for 10 minutes at room temperature covered in dark. This was followed by incubation with 50 µL of reagent B (0.1%, w/v, N-1-napthylethylenediamine dihydrochloride, Sigma-Aldrich) for 10 minutes at room temperature, protected from light, and A₅₄₃ nm was measured using a microplate reader. Serial dilutions of sodium nitrite (0–100 µM) were used to generate the nitrite standard curve.

RPMI 1640, fetal bovine serum (FBS), penicillin/streptomycin, amphotericin B, phosphate-buffered saline (PBS), Hank’s balanced salt solution (HBSS), and L-glutamine were purchased from Media Tech (Herndon, VA). Horse serum (HS) was obtained from Invitrogen (Waltham, MA). Cocaine-HCl (Ecgonine methyl ester benzoate, MW: 339.8), crystal violet, 2′,7′-dichlorofluorescin diacetate, L-glutaraldehyde, trypan blue, N-1-napthylethylenediamine dihydrochloride (NED), rhodamine (Rh)123 and EDTA (ethylene diamine tetraacetic acid) were supplied by Sigma Chemical Company (St. Louis, MO). All other routine chemicals were of analytical grade.