Nitric Oxide determination

We implemented the 2,3-diaminonaphthalene (DAN) assay to detect nitrites according to the method by Misko et al. (1993) and Kleinhenz et al. (2003) [64,65] with only minor modifications. In brief, DAN was dissolved in 0.62 N HCl at a concentration of 0.025 mg/ml. Supernatants from cultured cells were recovered and clarified by centrifugation at 400 xg for 10 minutes at 4°C. In order to avoid β-mercaptoethanol interference, co-culture supernatants were diluted 1:2 with RPMI1640 alone before measurement. Standard curves were prepared in matching media, i.e. DMEM + 10%FBS + L-Gln and Pen/Strep for pure MSC cultures or Complete Media:RPMI1640 (1:1) for co-culture experiments. Stock solution of nitrite was 20 mM in Mili-Q water, and it was diluted 1:100 in matching media to obtain a 200 μM working dilution, and then 1:10 as the first point of a two-fold serial dilution curve covering 10 data points. Dilution buffer and blanks were either DMEM- or RPMI-based media, depending on the corresponding experimental setting. Aliquots of sample supernatants and standard curves (100 μL) were placed into solid black Costar 3915 96-well plates (in triplicate), combined with DAN (20 μL) and incubated for 15 min at 37°C in the dark. After 15 min, 20 μL of 0.7 N NaOH was added to each well. Samples were analyzed for fluorescence using an Infinite M1000 plate reader (Tecan Group Ltd, Männedorf, Switzerland) using Fluorescence Top Reading. A full detailed protocol with detection settings and expected results is shown in S1 Protocol.