Spectra fluor plus instrument

The concentration of nitrite/nitrate was determined with the Saville-Griess assay adapted for microtiter plates. A standard curve was prepared with serial dilutions (0–50 µM) of a freshly prepared sodium nitrite (NaNO₂) stock solution (100 mM). Cells were homogenized in 1× PBS and, after centrifugation, 200 µl of the supernatant were added to a well of a 96-well plate. Fifty microliters of sulfanilamide solution (4 mg/ml in 1 N HCl) were added to standards and samples. After 2-min incubation, 50 μl of N-(naphtyl)-ethylenediamine dihydrochloride solution (6 mg/ml in H₂O) were added, followed by incubation for 5 min at room temperature and measuring absorbance at 540 nm with a Spectra Fluor Plus® instrument and XFluor® software (Tecan, Crailsheim).

The concentration of nitrite/nitrate was determined with the Saville‐Griess assay adapted for microtiter plates. A standard curve was prepared with serial dilutions (0‐50 μmol/L) of a freshly prepared sodium nitrite (NaNO₂) stock solution (100 mmol/L). Two hundred microlitre of the collected BMDM supernatants was added to a well of a 96‐well plate. Fifty microlitre of sulphanilamide solution (4 mg/mL in 1 N HCl) was added to standards and samples. After short incubation of 1‐2 minutes, 50 μL of N‐(naphthyl)‐ethylenediamine dihydrochloride solution (6 mg/mL in H₂O) was added followed by incubation for 5 minutes at room temperature and measuring absorbance at 540 nm with a Spectra Fluor Plus^® instrument and XFluor^® software (Tecan).