Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 502/535 |
MW | N/A |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | NeuroBIOLOGy ReactiveOxygenSpecies |
Related | CellSignaling BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1. Foradherentcells:Platecellsovernightingrowthmediumat20,000to80,000cells/well/90μLfora96-wellplateor5,000to20,000cells/well/22.5μLfora384-wellplate.
1.2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat80,000-200,000cells/well/90µLfora96-wellpoly-Dlysineplateor20,000-50,000cells/well/22.5µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareworkingsolution:
2.1 PrepareDAX-J2™PONGreenstocksolution(500X):Add20µLofDMSO(ComponentC)intothevialofDAX-J2™PONGreen(ComponentA),andmixthemwell.
Note:20μLofreconstitutedDAX-J2™PONGreenstocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat≤-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.2 PrepareDAX-J2™PONGreenworkingsolution(10X):Add10μLof500XDMSOreconstitutedDAX-J2™PeroxynitriteSensorstocksolution(fromStep2.1)into500μLofAssayBuffer(ComponentB),andmixthemwell.
Note:Theworkingsolutionisnotstable;prepareitasneededbeforeuse.
3.Runtheperoxynitriteassay:
3.1 Add10µL/well(96-wellplate),or2.5µL/well(384-wellplate)ofDAX-J2™PONGreenworkingsolution(fromStep2.2)in90µL(96-wellplate)or22.5µL(384-wellplate)cellcultureperwellinthecellplate(fromStep1).
Note:Itisnotnecessarytowashcellsbeforestaining.It’srecommendedtostainthecellsinfullmedium.
3.2 Co-incubatecellswithDAX-J2™PONGreenwithtestcompoundsinfullmediumorinyourdesiredbufferat37ºCfordesiredperiodoftime,protectedfromlight.Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
Note1:It’srecommendedtostainthecellsinfullmedium.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforestaining.Add90µL/well(96-wellplate)and22.5µL/well(384-wellplate)of1XHank’ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbestainedinserum-freemedia.
Note2:Weco-incubatedRAW264.7macrophagecellswith50-200μMSIN-1andDAX-J2™PONGreeninfullmediumat37ºCfor1hourtoinduceperoxynitrite.SeeFigure1fordetails.
3.3 Alternatively,staincellswithDAX-J2™PONGreenat37ºCfor1hour,protectedfromlight(asinStep3.1).Removetheworkingsolution,thentreatcellswithtestcompoundsinfullmediumorinyourdesiredbufferat37ºCfordesiredperiodoftime.
3.4 MonitorthefluorescenceincreaseusingmicroplatereaderatEx/Em=490/530nm(cutoff=515nm)withbottomreadmode,ortakeimagesusingfluorescencemicroscopewithaFITCfilter.
References&Citations | ![]() CitationExplorer |
Fluorescentreal-timequantitativemeasurementsofintracellularperoxynitritegenerationandinhibition
Authors:ZhenLuo,QinZhao,JixiangLiu,JinfangLiao,RuoguPeng,YuntingXi,ZhenjunDiwu
Journal:Analyticalbiochemistry(2017):44--48
GenesilencingofendothelialvonWillebrandFactorattenuatesangiotensinII-inducedendothelin-1expressioninporcineaorticendothelialcells
Authors:AnarDushpanova,SilviaAgostini,EnricaCiofini,ManuelaCABIati,ValentinaCasieri,MarcoMatteucci,SilviaDelRy,AldoClerico,SergioBerti,VincenzoLionetti
Journal:ScientificReports(2016)
AAT Bioquest常用产品报价单
产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |