CERTIFICATE OF ANALYSIS Nitric Oxide Assay Kit

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1 Ordering Code: EMSNO CERTIFICATE OF ANALYSIS Nitric Oxide Assay Kit Lot Number: MG Product: A complete kit for the quantitative determination of nitrite and nitrate in biological fluids (2 Plate, 96 Determination Kit). The kit uses the enzyme Reductase to convert nitrate to nitrite. Nitrite is then detected as a colored azo dye product of the Griess reaction that absorbs visible light at 540 nm. The interaction of nitric oxide (NO) in a system is measured by the determination of both nitrate and nitrite concentrations in the sample. The relative levels of nitrite and nitrate can vary substantially, therefore the most accurate determination of total NO production requires quantization of both nitrate and nitrite. Kit Storage: -20 C in a manual defrost freezer. Avoid repeated freeze-thaw cycles. NITRIC OXIDE KIT COMPONENTS **Allow all Kit Components to come to room temperature for at least 30 minutes prior to opening EXCEPT the Reductase enzyme. Description Size Form Component Usage Lot 10X Reagent Diluent Reductase Diluent Reductase 30 ml A HEPES based buffer containing detergent and 4 ml A phosphate based buffer containing Dilute 10 ml of the 10X Reagent Diluent with 90 ml of deionized water. This 1X Reagent Diluent can be stored at room temperature for 3 months. 1 vial Lyophilized Reductase Concentrate: Reconstitute with 1 ml of Reductase Diluent. Vortex vigorously. Let sit at room temperature for 15 minutes. Vortex. Let sit at room temperature for an additional 15 minutes. Vortex. Use on ice. Store tightly capped at -20 C in a manual defrost freezer. Stable at -20 C for 3 months. Avoid repeated freeze/thaw cycles. Diluted Reductase: Count the total number of wells needed for the samples plus 14 wells for the complete standard curve in duplicate. Use the following formula to calculate the volume of reconstituted Reductase Concentrate and 1X Reagent Diluent required for the Final Enzyme Dilution: A. Volume of reconstituted Reductase Concentrate (µl) = [Number of wells, including Standard Curve Wells + 2] x 10 µl B. Volume of 1X Reagent Diluent (µl) = [Volume Reconstituted Reductase Concentrate in µl (from A above)] x 1.5 µl Immediately before use, pipet the volume of reconstituted Reductase Concentrate from A. above and add to it the volume of 1X Reagent Diluent from B. above. Vortex and use on ice within 15 minutes. NADH 2 vials Lyophilized, reduced β-nicotinamide adenine dinucleotide. Store in the Dark. NADH Concentrate: Reconstitute by adding 1 ml of deionized water. Wait for 3 minutes and vortex prior to use. Use on ice. Diluted NADH: Prior to use take 0.9 ml of the NADH solution and add 1.8 ml of water. Vortex. Use on ice. For both the Concentrated and Diluted NADH: Use on ice. Store tightly capped at -20 C in a manual defrost freezer. Stable at -20 C for 45 days. Avoid repeated freeze/thaw cycles. MG157087E MG157087F MG157087B MG157087A

2 Nitrite Standard Standard Griess Reagent I Griess Reagent II Plate Sealers 0.5 ml A solution of sodium nitrite at 2,000 µm in water with 0.5 ml A solution of sodium nitrate at 1,000 µm in water with 12 ml A solution of sulfanilamide in 2 M HCl. 12 ml A solution of N-(1-Naphthyl) ethylenediamine in 2 M HCl. Label seven 12 x 75 mm glass Tubes #1 - #7. Pipet 900 µl of 1X Reagent Diluent into Tube #1. Pipet 500 µl of 1X Reagent Diluent into Tubes #2 - #7. Add 100 µl of Nitrite Standard (2,000 µm) to Tube #1 and vortex. Add 500 µl from Tube #1 to Tube #2 and vortex. Add 500 µl from Tube #2 to Tube #3 and vortex. Continue this for Tubes #4 - #7. The concentration of Nitrite in Tubes #1 - #7 will be 200, 100, 50, 25, 12.5, 6.25 and µm, respectively. Label seven 12x75 mm glass Tubes #1 - #6. Pipet 900 µl of 1X Reagent Diluent into Tube #1. Pipet 500 µl of 1X Reagent Diluent into Tubes #2 - #6. Add 100 µl of Standard (1,000 µm) to Tube #1 and vortex. Add 500 µl from Tube #1 to Tube #2 and vortex. Continue this for Tubes #3 - #6. The concentration of in Tubes #1 - #6 will be 100, 50, 25, 12.5, 6.25 and µm, respectively. 2 each N/A MG157087D MG157087C MG157087G MG157087H 1. Materials needed but not supplied with kit include: microwell plates; deionized or distilled water; precision pipets for volumes between 25 µl and 1,000 µl; repeater pipets for dispensing 25 µl and 50 µl; ice bath or refrigerated container capable of maintaining 0 C; a 37 C incubator; and a microplate reader capable of reading between nm. 2. CAUTION: Any sample or buffer component that may interfere with the Reductase enzyme will lower the conversion of sample nitrate to nitrite and therefore give rise to lower estimates of NO. Examples of nucleophiles and antioxidants that may interfere with the assays are azide, ascorbic acid, sulfhydryl containing compounds such as cysteine, glutathione, DTT and β-mercaptoethanol. If concentrations of these materials are to be in excess of 10 µm in the sample, a test of nitrate recovery should be made using the nitrate provided with the kit. at concentrations similar to those used for the standard curve should be added to the Procedural Notes buffer containing the suspected interfering compound and to a similar buffer without the suspected interfering compound. If there is a significant change, the effect should be determined and suitable corrections made. 3. Some tissue culture media, such as RPMI, contain high nitrate concentrations and should not be used, as this will interfere with sensitive detections. 4. Certain systems involving NO synthetase enzymes utilize high concentrations (0.5-1 mm) of NADPH, which may inhibit the Griess color reaction slightly. Care should be taken to ensure that these are diluted sufficiently ( 1:10) in 1X Reagent Diluent to minimize any effect of NADPH. If samples contain excessive amounts of NADPH, this can be oxidized using 10 µl of 1500 U/ml Lactate Dehydrogenase in 30 mm Na pyruvate after incubation with Reductase and incubated at 37 C for 10 minutes prior to addition of the Griess reagents.

3 Sample Handling Whole Organism Samples: Environmental nitrite and nitrate must be taken into account. Any media or fluid that the organism is stored in should be analyzed separately. Adjustments to the nitrite and nitrate in the organism must take into account the turnover of environmental nitrite and nitrate by the organism. Urine: Fresh urine samples can be diluted into 1X Reagent Diluent, ultrafiltered through a 10,000 molecular weight cut off (MWCO) filter and used directly in the assay. If the samples need to be stored, either suitable antibiotics, such as penicillin or streptomycin at 100 U/ml, or 2-propanol at 6.5% (v/v) can be added prior to storing at - 80 C. Saliva: Saliva samples can be diluted into 1X Reagent Diluent, ultrafiltered through a 10,000 MWCO filter and used directly in the assay. Typical saliva samples may contain a relatively high concentration of nitrate, which is thought to be produced by oral bacteria. : Citrate plasma is recommended. can be used directly in the assay after dilution into 1X Reagent Diluent and ultrafiltration through a 10,000 MWCO filter. EDTA or heparanized plasmas may be used in the assay after dilution into 1X Reagent Diluent and ultrafiltration through a 10,000 MWCO filter, however they may not give reproducible results as the protein may precipitate during the Griess reaction. Serum: Serum may be diluted into Reagent Diluent, ultrafiltered through a 10,000 MWCO filter and used directly in the assay. NITRITE ASSAY PROCEDURE All standards and samples should be run in duplicate. 1. Pipet 200 µl of 1X Reagent Diluent into duplicate Blank wells. 2. Pipet 50 µl of Nitrite standards #1 - #7 into duplicate wells. 3. Pipet 50 µl of the 1X Reagent Diluent into duplicate wells to act as a Zero Standard. 4. Pipet 50 µl of each Sample into duplicate wells. 5. Pipet 50 µl of the 1X Reagent Diluent into the Zero Standard, Standards #1 - #7 and Sample Wells. 6. Pipet 50 µl the Griess Reagent I into each 7. Pipet 50 µl the Griess Reagent II into each 8. Mix well by shaking or tapping the side of the plate. 9. Incubate the plate at room temperature (22 C - 25 C) for 10 minutes. 10.Read the optical density of each well at 540 ± 20 nm after blanking against the Blank Wells. NITRITE PLATE LAYOUT A Blank Blank Zero Zero B 1 1 C 2 2 D 3 3 E 4 4 F 5 5 G 6 6 H 7 7 Typical Standard Curve - Standard curves must be run with every assay

4 NITRATE ASSAY PROCEDURE Mix well and All standards and samples should be run in duplicate. 1. Pipet 200 µl of 1X Reagent Diluent into duplicate Blank wells. 2. Pipet 50 µl of Standards #1 - #6 into duplicate wells. 3. Pipet 50 µl of the 1X Reagent Diluent into duplicate wells to act as a Zero Standard. 4. Pipet 50 µl of each Sample into duplicate wells. 5. Pipet 25 µl of diluted NADH into all Standard and Sample Wells. 6. Pipet 25 µl of the Reductase Final Enzyme Dilution into all Standard and Sample Wells. 7. apply plate sealer to wells. Incubate for 30 minutes at 37 C. 8. Pipet 50 µl the Griess Reagent I into each 9. Pipet 50 µl the Griess Reagent II into each 10.Mix well by shaking or tapping the side of the plate. 11.Incubate the plate at room temperature (22 C - 25 C) for 10 minutes. 12.Read the optical density of each well at 540 ± 20 nm after blanking against the Blank Wells. NITRATE PLATE LAYOUT A Blank Blank B 1 1 C 2 2 D 3 3 E 4 4 F 5 5 G 6 6 H Zero Zero Typical Standard Curve - Standard curves must be run with every assay. Calculation of Results Several options are available for the calculation of the concentration of Nitrite or Total in the samples. 1. Calculate the average net Optical Density (OD) bound for each Standard and Sample by subtracting the average Zero Standard OD from the average OD for each Standard and Sample. Average Net OD = Average OD - Average Zero Standard OD 2. Plot the Average Net OD for each Standard versus Nitrite or Concentration. 3. Plot the Average OD for each Sample and extrapolate Nitrite or Total Nitrite and concentration from the graph. 4. Subtract the Nitrite concentration from the Total Nitrite and concentration to obtain the concentration in the sample. This kit allows the user to independently determine the concentrations of Nitrite and in the sample. Nitrite concentrations are determined from the Nitrite Standard Curve. concentrations are determined by conversion of the into Nitrite and measurement in the Nitrite assay. By subtracting the sample Nitrite concentration from the measured Nitrite concentration, after the enzymatic conversion of, the sample can be calculated. 1. Measure Nitrite Concentration in the Sample using Nitrite Assay Procedure = X µm 2. Measure Nitrite Concentration in Sample after conversion of into Nitrite using Assay Procedure = Y µm 3. Sample Nitrite Conc. = X µm 4. Sample Conc = Y - X µm 5. To calculate the concentration of Nitrite or in your sample, just multiply by the dilution factor used in preparing the samples.

5 PERFORMANCE CHARACTERISTICS The following parameters for this kit were determined using the guidelines listed in the National Committee for Clinical Laboratory Standards (NCCLS) Evaluation Protocols. Linearity A sample containing 125 µm Nitrite or 75 µm nitrate was diluted 6 times 1:2 in 1X Reagent Diluent and measured in the specific assay. The data was plotted graphically as actual concentration versus measured concentration. The line obtained for Nitrite had a slope of and a correlation coefficient of The line obtained for had a slope of and a correlation coefficient of Precision Intra-assay precision was determined by taking samples containing low, medium and high concentrations of Nitrite and and running these samples multiple times (n=8) in the same assay. Inter-assay precision was determined by measuring them in multiple assays (n=8). The precision numbers listed below represent the percent coefficient of variation for the concentrations of Nitrite and determined in these assays as calculated by a linear curve fitting program. Intra-assay Nitrite (µm) %CV (µm) %CV Low Medium High Inter-assay Nitrite (µm) %CV (µm) %CV Low Medium High Sensitivity Sensitivity was calculated by determining the average OD for 16 wells run as Zero Standard, and comparing to the average OD for 16 wells run with Standard #7 for Nitrite and 16 wells run with Standard #6 for. The detection limit was determined as the concentration of Nitrite or measured at 2 standard deviations from the zero along the standard curve. Nitrite Sensitivity = µm Sensitivity = µm Sample Recoveries Nitrite and concentrations were measured in a variety of different samples, which had been diluted with 1X Reagent Diluent. The diluted samples were ultrafiltered through a 10,000 MWCO filter and assayed in the kit. Nitrite Assay Assay* Sample % Recovery Rec. Dilution % Recovery Rec. Dilution Cell Culture 100 1:2 82 1:2-1:10 Media Saliva 96 1: :2-1:100 Urine 110 1:2 87 1:2-0 Serum 112 1:2 98 1:2 - Citrate EDTA Heparin 118 1:2 98 1:2-89 1:2 86 1: :2 96 1:2 - *Please note that the recommended dilutions for the Assay take into account the normal levels of in some samples. Quality Control Signature documentation, specifications and/or accompanying package inserts ( Documentation ) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. Buyer s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s). There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not

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