Sulfanilamide

For quantification of nitrate, sulfanilamide (AppliChem GmbH, Darmstadt, Germany), ammonium chloride and hydrochloric (Th. Geyer, Renningen, Germany), sodium nitrite and ammonia solution 25% (Merck, Darmstadt, Germany), and N-(1-naphthyl)-ethylene diamine dihydrochloride (Carl Roth GmbH, Karlsruhe, Germany) were used. Regarding total phenolic content measurement, Folin–Ciocalteu reagent and gallic acid were provided by Merck (Darmstadt, Germany). Na₂CO₃ and methanol were purchased from AppliChem GmbH (Darmstadt, Germany) and Carl Roth GmbH (Karlsruhe, Germany), respectively. Ethanol needed for betalain analysis was purchased from Th. Geyer (Renningen, Germany).

Kynurenine (Kyn) and nitrite concentrations were measured in co-culture SN of 3-day co-cultures under stimulated or un- stimulated conditions.
For Kyn, 100 µl of standard (50 mM L-kynurenine, Santa Cruz Biotechnology, Heidelberg, Germany) or sample was added to 50 µl of 30% trichloroacetic acid (Carl Roth GmbH, Karlsruhe, Germany). The samples were incubated for 30 minutes at 50°C, then centrifuged (10 minutes at 4,000 x g) and then 75 µl of the supernatant was mixed with 75 µl 2% 4-(Dimethylamino) benzaldehyde (Santa Cruz Biotechnology). After 15 minutes at RT, the optical density (OD) was measured (TECAN infinite M200PRO, Tecan Deutschland GmbH, Crailsheim, Germany) at 492 nm emission. Standard curves were constructed using GraphPad Prism 9.1.0 Software (GraphPad Software, California, USA).
For nitrite measurements, 150 µl of standard sodium nitrite (0.1 µmol/ml sodium nitrite dissolved in RPMI, Applichem, Darmstadt, Germany) or samples were diluted in RPMI 1640 media, mixed with 50µl of sulfanilamide (AppliChem) and incubated for 2 minutes. 50 µl of N-(1-Napthyl)-ethylendiamine dihydrochloride (naphtylamine) (Carl Roth GmbH) was added before another incubation for 30 minutes at RT in the dark. The OD was measured using 542 nm emission/620 nm. Standard curves were constructed using GraphPad Prism 9.1.0 Software and the limit of detection was calculated.

Nitrite (NO₂₋) was detected in CM from IDO assay or cocultures (human and rat) and controls of stimulated and not stimulated conditions. A total of 150 µL of probes or standard dilutions (0.1 µmol/mL sodium nitrite dissolved in RPMI) were mixed with sulfanilamide (both AppliChem) in a 96-well clear plate. Plate was incubated during two minutes before the addition of N-(1-napthyl)-ethylendiamine dihydrochloride (naphtylamine) (Carl Roth GmbH), then incubated for 30 min in the dark at RT. OD of each well was determined using a microplate reader using 542 nm emission wavelength and 620 nm reference wavelength. Standard curves were elaborated with GraphPad Prism 7 software and limit of detection was calculated and applied to measured values.