To quantify NO via the Griess assay,41 50 μL of a 2 mg mL-1 solution of PROLI/NO in 100 mM sodium hydroxide (NaOH) was added to 15 mL of desired media and incubated at room temperature for at least 24 h. Aliquots (50 μL) of this sample were added to a sulfanilamide solution (50 μL) and incubated in the dark at room temperature for 5 min. Naphthylethylenediamine (50 μL) was added to the mixture to form a colorimetric product with concomitant absorbance measured in each well at 540 nm using a LabSystems MultiSkan RC microplate reader (Helsinki, Finland). Sodium nitrite standards were used to normalize the assay reactivity and associated absorbance.
For analysis of blood constituents (i.e., plasma and serum), NADPH (25 μL) and nitrate reductase (2 μL) were added to the samples and allowed to incubate for at least 30 min prior to the addition of the Griess reagents. Headspace studies using Griess were conducted in the same manner, but the volume of media added to the 20 mL scintillation vial was varied (10, 15, and 20 mL). Concentration dependence studies were conducted by varying the concentration of the stock PROLI/NO solution and injecting aliquots into 15 mL media to yield final PROLI/NO concentrations of 0.67, 6.7, and 67 μg mL-1.
For analysis of blood constituents (i.e., plasma and serum), NADPH (25 μL) and nitrate reductase (2 μL) were added to the samples and allowed to incubate for at least 30 min prior to the addition of the Griess reagents. Headspace studies using Griess were conducted in the same manner, but the volume of media added to the 20 mL scintillation vial was varied (10, 15, and 20 mL). Concentration dependence studies were conducted by varying the concentration of the stock PROLI/NO solution and injecting aliquots into 15 mL media to yield final PROLI/NO concentrations of 0.67, 6.7, and 67 μg mL-1.
