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For nitrite only: 50μL, For Nitrite/nitrate: 100μL
Reagent Preparation: 1. To prepare Reagent A solution: Add exactly 1000 μL of ultrapure water to Reagent A and mix thoroughly. This reagent must be freshly prepared. 2. To prepare Reagent C solution: Add exactly 1000 μL of ultrapure water to Reagent C and mix thoroughly. This reagent must be freshly prepared.
It is recommended to assay the samples in duplicate. Plasma samples may be deproteinized before performing the assay.
Standard Preparation: Prepare the calibrate in 1 mL tubes following the Table 1 (see kit booklet). Use ultrapure water as diluent. Performing the assay: The following procedure is for the determination of nitrite + nitrate: 1. Add 50 μL of the sample or standard in each well (96-well plate). 2.Add 10 μL of Reagent A and 20 μL of Reagent B. Incubate for 60 minutes. 3.Add 10 μL of Reagent C and 10 μL of Reagent D. Incubate for 20 minutes. 4.Add 50 μL of Reagent E in each well. Incubate for 10 minutes protected from light. 5.Add 50 μL of Reagent F in each well. Incubate for 10 minutes protected from light. 6.Read the absorbance at 540 nm within 30 minutes. In order to measure only the nitrite in the sample (not both nitrite and nitrate, "total nitrite"), add ultrapure water instead of Reagents A, B, C and D and continue the assay by adding Reagents E and F as shown in the procedure.
1.Calculate the average absorbance of each sample and control. 2.Determine the concentration in the sample by comparison to the Nitrite Standard reference curve (Figure 4 see Images). Nitrite (μM) = (?A540nm- intercept)/ slope