Part I. In vitro determination of nitrite concentration

Materials and Reagents

1. Square Petri dishes (Deltalab, catalog number: 200204 )
   
2. Multi-well plates (96 well) (Deltalab, catalog number: 900010 )
   
3. 10-day-old Arabidopsis ecotype Columbia Col-0
   
4. Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524 )
   
5. Sodium choride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
6. Sulfanilamide (Sigma-Aldrich, catalog number: S9251 )
   Note: The working solution is 1% (w/v) Sulfanilamide in 5% (v/v) phosphoric acid. Store at 4 ºC in the dark.
   
7. N-(1-Naphthyl)ethylenediamine dihydrochloride (NED) (Sigma-Aldrich, catalog number: 33461 )
   Note: The working solution is 0.1% (w/v) NED in H₂O. Store at 4 ºC in the dark.
   
8. Standard nitrite solution (Sigma-Aldrich, catalog number: 237213 )
   Note: The working solution is 100 µM sodium nitrite in Milli Q water.
   
9. Sodium phosphate dibasic (Sigma-Aldrich, catalog number: S0876 )
   
10. Sodium phosphate monobasic (Sigma-Aldrich, catalog number: 0751 )
    
11. Buffer A (100 mM phosphate buffer, pH 7.4) (see Recipes)
    

Equipment

1. Centrifuge (Thermo Fisher Scientific, model: Sorvall Legend Micro 17R )
   
2. Elisa plate reader (Metrolab 980 microplate reader)
   

Procedure