Total Nitric Oxide Assay Kit

Transcription

1 Ttal Nitric Oxide Assay Kit Catalg Number: EMSNOTOT Prduct descriptin A cmplete kit fr the quantitative determinatin f ttal nitric xide (NO) in bilgical fluids. The kit uses the enzyme nitrate reductase t cnvert nitrate t nitrite. Nitrite is then detected as a clred az dye prduct f the Griess reactin that absrbs visible light at 540 nm. The interactin f NO in a system is measured by the determinatin f ttal nitrate and nitrite cncentratins in the sample. This kit allws fr the ttal determinatin f bth NO prducts in the sample by cnversin f all f the sample nitrate t nitrite, fllwed by the determinatin f the ttal cncentratin f nitrite in the sample. Cntents and strage Upn receipt, stre the kit at 20 C. Avid repeated freeze-thaw cycles. Descriptin 10X Reagent Diluent Nitrate Reductase Diluent Nitrate Reductase NADH, light-sensitive Nitrate Standard (1,000 µm) Griess Reagent I Griess Reagent II Size 30 ml 4 ml 2 vials 2 vials 0.5 ml 12 ml 12 ml Additinal required materials 96-well micrtiter plates and plate sealers Deinized r distilled water. Precisin pipettes (fr vlumes between 25 µl and 1,000 µl). Repeater pipettes (fr dispensing 25 µl and 50 µl). Dispsable beakers fr diluting buffer cncentrates mm glass tubes. Graduated cylinders. Ice bath r refrigerated cntainer capable f maintaining 0 C. 37 C incubatr. Micrplate reader capable f reading between nm. 10,000 MWCO plysulfne filters. General guidelines D nt mix cmpnents frm different kit lts r use reagents beynd the kit expiratin date. Sample r buffer cmpnent that may interfere with the enzyme will lwer the cnversin f sample nitrate t nitrite and give rise t lwer estimates f NO. Nuclephiles and antixidants that may interfere with the assays include azide, ascrbic acid, and sulfhydryl cntaining cmpunds such as β-mercaptethanl, cysteine, DTT, and glutathine. Test nitrate recvery using the nitrate prvided with the kit if cncentratins f these materials are >10 µm in the sample. Add nitrate at cncentratins similar t thse used fr the standard curve t the buffer cntaining the suspected interfering cmpund and cmpare results t a similar buffer withut the suspected interfering cmpund. If there is a significant change, make the apprpriate crrectins. D nt use samples in tissue culture media with high nitrate cncentratins (e.g. RPMI). Allw kit cmpnents except the Nitrate Reductase enzyme t cme t rm temperature fr at least 30 minutes befre use. Fr Research Use Only. Nt fr use in diagnstic prcedures. Manufacturing site: Bender MedSystems GmbH Campus Vienna Bicenter Vienna, Austria

2 Prepare 1X Reagent Diluent Dilute 10 ml f 10X Reagent Diluent with 90 ml f deinized water. Label as 1X Reagent Diluent. The diluted buffer is stable fr up t 3 mnths at rm temperature. Recnstitute and dilute NADH reagent 1. Add 1 ml f deinized water t a vial f NADH. 2. After 3 minutes, vrtex prir t use. Use n ice. 3. Dilute 0.9 ml f NADH slutin with 1.8 ml f deinized water in a new tube placed n ice. 4. Use diluted NADH slutin n ice. 5. Stre diluted and undiluted NADH slutin tightly capped at 20 C in a manual defrst freezer. The slutins are stable fr 45 days at 20 C. Resuspend Nitrate Reductase Cncentrate 6. Add 1 ml f Nitrate Reductase Diluent t a vial f Nitrate Reductase Cncentrate. 7. Vrtex vigrusly and let sit at rm temperature fr 15 minutes. 8. Vrtex again and let sit at rm temperature fr an additinal 15 minutes. 9. Use enzyme n ice and stre tightly capped at 20 C in a manual defrst freezer. Avid repeated freeze-thaw cycles. The diluted enzyme is stable fr up t 3 mnths at 20 C. Prepare Diluted Nitrate Reductase 1. Determine the number f wells needed fr samples and add 14 wells fr the cmplete standard curve (in duplicate). 2. Calculate the required vlume f recnstituted Nitrate Reductase Cncentrate using the fllwing frmula: Vlume f Nitrate Reductase Cncentrate (µl) = [Ttal number wells fr samples and standards + 2] 10 µl 3. Calculate the required vlume f 1X Reagent Diluent using the fllwing frmula: Vlume f 1X Reagent Diluent (µl) = [Vlume Nitrate Reductase Cncentrate (step 2)] 1.5 µl 4. Add 1X Reagent Diluent (step 3) t a clean tube, and place n ice. 5. Immediately befre use, add Nitrate Reductase Cncentrate (step 2) t the tube cntaining 1X Reagent Diluent. 6. Vrtex the tube and use n ice within 15 minutes. Whle Organism Samples Take envirnmental nitrite and nitrate levels int accunt. Adjustments t the nitrite and nitrate levels in the sample must take int accunt the turnver f envirnmental nitrite and nitrate by the rganism. Analyze media r fluid that the rganism is stred in separately. Urine Fresh urine samples shuld be diluted at least 1:20 with 1X Reagent Diluent, and ultrafiltered thrugh a 10,000 mlecular weight cut ff (MWCO) filter. Add antibitics (e.g. penicillin r streptmycin at 100 U/mL), r 2- prpanl at 6.5% (v/v) t the samples, and freeze at 80 C if samples will nt be tested immediately. Saliva Saliva samples shuld be diluted 1:2 1:100 int 1X Reagent Diluent, and ultrafiltered thrugh a 10,000 mlecular weight cut ff (MWCO) filter. Typical saliva samples may cntain relatively high cncentratins f nitrate prduced by ral bacteria. Plasma Plasma shuld be diluted 1:2 1:20 in 1X Reagent Diluent and ultrafiltered thrugh a 10,000 MWCO filter. Citrate plasma is recmmended. EDTA r heparinized plasma may be used, but may nt give reprducible results as the prtein may precipitate during the Griess reactin. Serum Serum samples shuld be diluted 1:2 1:20 in 1X Reagent Diluent, and ultrafiltered thrugh a 10,000 mlecular weight cut ff (MWCO) filter. Culture Supernatant Avid media cntaining nitrates. Samples shuld be diluted at least 1:2 in 1X Reagent Diluent, and ultrafiltered thrugh a 10,000 mlecular weight cut ff (MWCO) filter. Sample preparatin guidelines All samples shuld be diluted at least 1:2 with 1X Reagent Diluent. NO synthetase enzyme systems using high cncentratins (0.5 1 mm) f NADPH, may inhibit the Griess clr reactin slightly. Dilute such samples are sufficiently ( 1:10) in 1X Reagent Diluent t minimize the effects f NADPH. Oxidize NADPH with 10 µl f lactate dehydrgenase (1500 U/mL in 30 mm sdium pyruvate) after incubatin with nitrate reductase (See Nitrate assay prcedure, step 8), and incubate at 37 C fr 10 minutes befre adding the Griess reagents. Fr Research Use Only. Nt fr use in diagnstic prcedures. Manufacturing site: Bender MedSystems GmbH Campus Vienna Bicenter Vienna, Austria

3 Dilute standards Allw the Nitrate Standard slutin t warm t rm temperature befre use. Label six mm glass tubes #1 thrugh #6. Add 900 μl 1X Reagent Diluent t Tube #1. Add 500 μl 1X Reagent Diluent t Tubes #2 t #6. Add 100 μl Nitrate Standard t Tube #1 and vrtex thrughly. Add 500 μl f Tube #1 t Tube #2 and vrtex thrughly. Cntinue t make serial dilutins f the standard as shwn in the diagram belw. Calculatins Analyze the data with a 4-parameter lgistic curve-fitting prgram. If data reductin sftware is nt available, the cncentratin f ttal NO can be calculated by the fllwing methd: 1. Calculate the average net Optical Density (OD) bund fr each standard and sample by subtracting the average Zer Standard OD frm the average OD fr each standard and sample. Average Net OD = Average OD Average Zer Standard OD 2. Plt the Average Net OD fr each Standard Cncentratin. 3. Plt the Average OD fr each Sample and interplate Ttal NO Cncentratin frm the graph. Nitrate assay prcedure Determine the number wells required fr the assay. Bring all reagents t rm temperature fr at least 30 minutes befre pening. Run all standards and samples in duplicate. 1. Add 200 μl f 1X Reagent Diluent int the Blank 2. Add 50 μl f Nitrate Standards #1 thrugh #6 int the apprpriate 3. Add 50 μl f 1X Reagent Diluent int the Zer Standard 4. Add 50 μl f the Samples int the apprpriate 5. Add 25 μl f diluted NADH int all Zer Standard, Standard, and Sample 6. Add 25 μl f Diluted Nitrate Reductase int all Zer Standard, Standard, and Sample 7. Tap the plate gently t mix the cntents. 8. Seal the plate and incubate at 37 C fr 30 minutes. 9. Add 50 μl f Griess Reagent I int each well, except the Blank 10. Add 50 μl f Griess Reagent II int each well, except the Blank 11. Tap the plate gently t mix the cntents. 12. Incubate the plate at rm temperature fr 10 minutes. 13. Blank the plate reader against the Blank wells, and read the ptical density at nm. If the plate reader is nt able t be blanked against the Blank wells, manually subtract the mean ptical density f the Blank wells frm all readings. Fr Research Use Only. Nt fr use in diagnstic prcedures. Manufacturing site: Bender MedSystems GmbH Campus Vienna Bicenter Vienna, Austria

4 Typical standard curve A typical standard curve is shwn belw. This curve must nt be used t calculate nitrate cncentratins; a standard curve must be run with every assay. Precisin Intra-assay precisin was determined by taking samples cntaining lw, medium and high cncentratins f nitrate and running these samples multiple times (n=8) in the same assay. Inter-assay precisin was determined by measuring 3 samples with lw, medium and high cncentratins f ttal nitrate in multiple assays (n=8). The precisin numbers listed belw represent the percent cefficient f variatin fr the cncentratins f nitrate determined in these assays as calculated by a 4-parameter lgistic curve-fitting prgram. Intra-assay Nitrate (µm) %CV Lw % Medium % High % Inter-assay Nitrate (µm) %CV Lw % Medium % Perfrmance characteristics The fllwing parameters fr this kit were determined using the guidelines listed in the Natinal Cmmittee fr Clinical Labratry Standards (NCCLS) Evaluatin Prtcls. Sensitivity The minimum detectable dse f nitrate is µm. This was determined by adding tw standard deviatins t the mean O.D. btained frm the average OD bund fr 24 wells run as Zer Standard, cmpared t the average OD fr wells run with Standard #6. Linearity A sample cntaining 75 µm nitrate was serially diluted in 1X Reagent Diluent ver the range f the assay. Linear regressin analysis f samples versus the expected cncentratin yielded a line with a slpe f and crrelatin cefficient f High % Sample recvery Nitrate was spiked int samples which had been diluted with 1X Reagent Diluent. The diluted samples were ultrafiltered thrugh a 10,000 MWCO filter and assayed in the kit. The fllwing results were btained. Sample % Recvery Recmmended Dilutin Cell Culture Media 88% 1:2 Human Saliva 100% 1:2 t 1:100 Human Urine 104% 1:20 Human Serum 93% 1:2 t 1:20 Human Citrate Plasma 98% 1:2 t 1:20 Human EDTA Plasma 91% 1:2 t 1:10 The recmmended dilutins fr the assay take int accunt the nrmal nitrate levels in sme samples. All samples are diluted 1:2 in 1X Reagent Diluent, but sme samples need t be diluted 1:2 t 1:200 t read n the Nitrate Standard Curve. Limited prduct warranty Fr Research Use Only. Nt fr use in diagnstic prcedures. Manufacturing site: Bender MedSystems GmbH Campus Vienna Bicenter Vienna, Austria

5 Life Technlgies Crpratin and/r its affiliate(s) warrant their prducts as set frth in the Life Technlgies' General Terms and Cnditins f Sale fund n Life Technlgies' website at If yu have any questins, please cntact Life Technlgies at Manufacturer's address: Bender MedSystems GmbH Campus Vienna Bicenter Vienna, Austria The infrmatin in this guide is subject t change withut ntice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Imprtant Licensing Infrmatin: These prducts may be cvered by ne r mre Limited Use Label Licenses. By use f these prducts, yu accept the terms and cnditins f all applicable Limited Use Label Licenses Therm Fisher Scientific Inc. All rights reserved. All trademarks are the prperty f Therm Fisher Scientific and its subsidiaries unless therwise specified. Fr Research Use Only. Nt fr use in diagnstic prcedures. Manufacturing site: Bender MedSystems GmbH Campus Vienna Bicenter Vienna, Austria