Ng methyl l arginine acetate salt l nmma

CD4⁺T cells were labeled with 0.5 μmol/L CFSE. Subsequently, the cells were washed and co-cultured at different ratios with M-MDSCs (16:1, 4:1, 1:1) and CD14⁺HLA-DR⁺cells (1:1) in 96-well plates in complete RPMI 1640 medium with 10% fetal bovine serum. CD4⁺T cells cultured alone were used as positive control. All cells were incubated with anti-CD3 (2 μg/mL), anti-CD28 (5 μg/mL). After 3 days of co-culture, the proliferation of CD4⁺T cells was determined by FACS analysis. Additionally, the inhibitors of candidate suppressive molecules were added at the following final concentrations in the co-culture of CD4⁺T cells and M-MDSCs (1:1): 500 μmol/L Nx-Hydroxy-nor-L-arginine, diacetate salt (nor-NOHA, Calbiochem, Darmstadt, Germany), and 500 μmol/L NG-Methyl-L-arginine acetate salt (L-NMMA, Sigma-Aldrich). Supernatants were stored at −80°C until further use. The concentration of IFN-γ was measured by ELISA Kit according to the manufacturer's instructions.

The cells were stimulated in the absence or presence of either IFN-γ (10 ng/ml) (Sigma-Aldrich, Taufkirchen, Germany) and Lipopolysaccharide (LPS) (50 ng/ml) (Sigma-Aldrich) in presence or absence of N^G-Methyl-L-arginine acetate salt (L-NMMA) (1 mg/ml) (Sigma-Aldrich). The cells were also stimulated with SLAs (10 μg/ml or 20 μg/ml). Supernatants were collected 24 and 48 h post-stimulation and stored at -20 °C until use for nitrite assay.
Nitrite (NO₂⁻) is a stable compound produced by the reaction of NO with water and oxygen, and its accumulation reflects the amount of NO produced in culture medium. NO₂⁻ assay was performed according to Griess protocol previously described [20 (link)]. Briefly, 100 μl of supernatants were distributed to 96-well plates in triplicates and 100 μl of Griess reagent (1% sulfanilamide, 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride and 2.5% H₃PO₄) (Sigma-Aldrich) was added. The plates were incubated at room temperature and optical density (OD) was read at 540 nm using a BioRad Micro-plate Reader Photometer (BioRad Dynex, Washington, USA). The NO concentration was calculated from a standard curve generated with NaNO₂ ranging from 0 to 200 μM. The experiments were performed in triplicates and the results represent the average of 6 independent experiments.

Sodium acetate, lipopolysaccharides (LPS) from Escherichia coli O111:B4, nigericin sodium salt, potassium chloride (KCl), 4-[[4-Oxo-2-thioxo-3-[[3-(trifluoromethyl)phenyl]methyl]-5-thiazolidinylidene]methyl]benzoic acid (CY-09), 2-deoxy-D-glucose (2DG), Syrosingopine, Etomoxir and NG-Methyl-L-arginine acetate salt (L-NMMA) were purchased from Sigma (Saint Louis, MO). IL-1β and IL-18 recombinant proteins were purchased from Invitrogen (Waltham, MA). anti-IL-1β neutralizing monoclonal antibody was purchased from Thermo Fisher Scientifics (Waltham, MA). Rabbit monoclonal antibody anti-ACSS2 (D19C6) was purchased from Cell Signaling (Danvers, MA), mouse monoclonal antibody anti-caspase-1 (Casper-1) was purchased from Adipogen (San Diego, CA). Rabbit polyclonal antibody anti-IL-1β was a kind gift from Proteintech (Rosemont, IL). Mouse IgG kappa binding protein conjugated to horseradish peroxidase (HRP) and mouse anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Dallas, TX).

Production of NO in hPOD and ciGEnC was visualized using the NO-sensitive dye 4-Amino-5-Methylamino-2′,7′-Difluoroscein Diacetate (DAF-FM diacetate, Thermofisher Scientific, Breda, The Netherlands). Differentiated cells were incubated for 60 min with 10 µM DAF-FM diacetate in phenol red-free (Thermofisher Scientific, Breda, The Netherlands) and fetal bovine serum (FBS)-free medium at 37 °C. Cells were subsequently washed three times with Hank’s balanced salt solution (HBSS, Gibco, Breda, The Netherlands) to remove excess probe and rested for an additional 45 min to allow the complete de-esterification of the probe. Where indicated, cells were pre-treated with 2 mM of the NOS inhibitor NG-Methyl-l-arginine acetate salt (L-NMMA, Sigma-Aldrich, Schnelldorf, Germany). Cell nuclei were stained with 1 µg/mL Hoechst 33342 (Thermofisher Scientific, Breda, The Netherlands) for 5 min. Three independent experiments were performed, and each experiment consisted of at least two samples. At least five fluorescent images were captured using a Zeiss Imager (Carl Zeiss Microscopy, Jena, Germany). An M1 microscope was used for each sample immediately upon completing the staining. FIJI (1.47v, National Institutes of Health, Bethesda, Rockville, MD, USA) was used for the quantification of fluorescent intensity stainings. The experiment was repeated in triplicate.

As reported previously [31 (link), 32 (link)], murine macrophage cell line RAW264.7 obtained from Cell Bank of Chinese Academy of Sciences (Beijing, People’s Republic of China), were seeded in 96-well cell culture plates (1.5 × 10⁵ cells/well) and treated with serial dilutions of the compounds with a maximum concentration of 50 μM in triplicate, followed by stimulation with 1 μg/mL LPS (Sigma, St. Louis, MO, USA) for 18 h. Nitric oxide production in the supernatant was assessed by Griess reagents (Reagent A & Reagent B, respectively, Sigma). The absorbance at 570 nm was measured with a microplate reader (Thermo, Waltham, MA, USA). NG-Methyl-l-arginine acetate salt (L-NMMA, Sigma), a well-known nitric oxide synthase (NOS) inhibitor, was used as a positive control. All the compounds were prepared as stock solutions in DMSO. The viability of RAW264.7 cells was evaluated by the MTS assay simultaneously to exclude the interference of the cytotoxicity of the test compounds. Concentration of a compound inhibiting 50% of cell growth (IC₅₀) was calculated by the Reed and Muench method.