1. Dissolve 278 mg FeSO₄, 7H₂O (ferrous sulfate heptahydrate) with 20 ml water^a) to prepare 50 mM FeSO₄ solution.^b)
2. Dissolve 123 mg DTCS Na with 10 ml watera) to prepare 50 mM DTCS solution.
3. Mix 1 ml DTCS Na solution with 8.8 ml buffer solutiona) (pH 7 or higher). Add 200 μl FeSO₄ solution just prior to use.c)
a) Purge any dissolved oxygen in the water or the buffer by nitrogen gas bubbling for at least 30 minutes prior to dissolving FeSO₄.
b) The FeSO₄ solution can be stored at -20ºC for at least 2 months.
c) Fe(II)-DTCS complex is colorless. If the solution is brown, Fe(III)-DTCS may have formed because of dissolved oxygen in the solution. However, the brown solution can still be used for NO trapping.
Preparation of NO-Fe(II)-DTCS Complex
1. Under argon gas flow, add 200 μl of FeSO₄ solution to 9.4 ml buffer solution (pH 7 or higher). Then introduce NO into the solution by bubbling NO gas through a glass capillary for 15 minutes.
2. Add 400 μl of DTCS Na solution to the FeSO₄ solution, and continue to introduce NO by bubbling for another 5 minutes.
3. Remove excess NO with argon gas bubbling for 5 minutes, and store at -20ºC. The NO-Fe(II)-DTCS solution can be stored at -20ºC for at least 2 months in oxygen-free conditions.