GE Analytical Instruments ©2006 12-5 DLM 14291 Rev. A
Preparation of Nitrite Standard Solutions
An analytical balance capable of weighing to ± 0.1 mg, a supply of reagent grade
sodium or potassium nitrite, nitrite-free, deionized water, volumetric flasks and
pipettes are required to calibrate the NOA for nitrite measurements.
Preparation of Stock Solution
A standard solution of ~100 mM NO
2
-
is prepared and dilutions of the standard used
for constructing the calibration curve.
To prepare 10 mL of 100 mM NO
2
-
:
• Weigh out ~69 mg of NaNO
2
or ~85 mg of KNO
2
directly into a 10 mL volumetric
flask.
• Record the weight of NaNO
2
or KNO
2
.
• Dilute to the mark with nitrite-free, deionized water.
• Invert the flask several times until the solid dissolves.
• Transfer the stock solution to air-tight storage container.
• Calculate the concentration of the stock solution. For NaNO
2
, the concentration
is
Conc. of Stock = (mgNaNO
2
/69 mg/mmol)/10 mL x 1000 mL/L
Use this stock 100 mM standard to prepare dilutions to construct a standard curve
for the NOA. The concentrations of the dilutions will depend on the nitrite levels
in the samples. Prepare dilutions that contain nitrite at concentrations greater
than the samples, concentrations less than the samples and concentrations
approximately the same as the samples. For most cell culture work, nitrite
concentrations will be in the nanomolar to micromolar range. Analysis of standard
solutions containing 10 nM, 50 nM, 100 nM, 1 µM, 5 µM, 10 µM, 50 µM and 100 µM
will allow construction of a calibration curve suitable for most cell culture work.
For stimulated macrophages and other cell types that produce high levels of NO,
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