| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 630/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | IonChannels ChlorideChannels |
| Related | pHandIonIndicators BiochemicalAssays |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinpre-warmedassaybufferat125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareiodideassayreagents:
2.1 Warmallthereagentstoroomtemperaturebeforeuse.
2.2 Make1XI-sensorenhancersolution:Add50µLof100XIodidesensorenhancer(ComponentB)to5mLofsterileH2O,andmixthemwell.
Note1:1XI-sensorenhancersolutionisnotstable,usewithin2hoursafterthedilution.
Note2:EachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimaldilutionofI-sensorenhancersolution.Wenoticedthat0.1XI-sensorenhancersolutionworksevenbetterforsomecelllines.
2.3 Make1Xcelllysisbuffer:Addthewholevialof10XCellLysisBuffer(ComponentD)to45mLofsterileH2O,andmixthemwell.
Note:5mLof1Xcelllysisbufferisenoughforoneplate.Storeunused1Xcelllysisbufferat4oC.
3.Foriodideeffluxassay:
3.1 Aspiratethegrowthmediumfromthecellplate.
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofpre-warmedI-LoADIngBuffer(ComponentC)andincubatefor2-4hours.
3.3 Aspiratetheiodideloadingbuffercompletely,andwashthecellswithDPBSorHBSSatleast3times.
3.4 TreatthecellswithagoNISTinHBSSbufferfor5minutes.
Note:Forantagonistsscreen,incubatethecompoundswithI-loadingbufferforatleastanadditional30minbeforethecellswerewashedwithDPBSorHBSSbuffer.
3.5 Aspiratethesupernatant.
3.6 Lysethecellsbyadding50µL/well(96-wellplate),or25µL/well(384-wellplate)of1Xcelllysisbuffer(fromStep2.3).
3.7 Performtheiodideassay(SeeStep5).
4.Foriodideinfluxassay:
4.1 Aspiratethegrowthmediumfromthecellplate.
4.2 Add100µL/well(96-wellplate),or25µL/well(384-wellplate)ofpre-warmedI-LoadingBuffer(ComponentC)withtestcompounds,andincubatefor5minutes.
4.3 Aspiratetheiodideloadingbuffercompletely,andwashthecellswithHBSS3times.
4.4 Lysethecellsbyadding50µL/well(96-wellplate),or25µL/well(384-wellplate)of1Xcelllysisbuffer(fromStep2.3)
4.5 Performtheiodideassay(SeeStep5).
5.Runiodideassay:
5.1 Add50µL/well(96-wellplate),or25µL/well(384-wellplate)ofIodideBlue™sensor(ComponentA)tothewellsthatcontaindifferentconcentrationsofpotassiumiodide(fromStep3.7orStep4.5).
5.2 Add50µL/well(96-wellplate),or25µL/well(384-wellplate)of1Xiodidesensorenhancersolution(fromStep2.2)intothemixture(Step5.1).
Note:Forsomecelllines,youmightneedtodiluteenhancersolutiondownto0.1X.
5.3 Incubateatroomtemperaturefor10sec-10min.
Note1:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalincubationtime.
Note2:Thebluecolormaychangetoyellowwithinsecondstominutesduetothepresenceofahighconcentrationofiodide.
5.4 Monitorabsorbanceat630,380,or405nm.
| References&Citations |  PrinterFriendlyVersion |
1. BregestovskiP,WaseemT,MukhtarovM.(2009)Geneticallyencodedopticalsensorsformonitoringofintracellularchlorideandchloride-selectivechannelactivity.FrontMolNeurosci,2,15.
2. CaputoA,HinzpeterA,CaciE,PedemonteN,ArousN,DiDucaM,Zegarra-MoranO,FanenP,GaliettaLJ.(2009)Mutation-specificpotencyandefficacyofcysticfibrosistransmembraneconductanceregulatorchloridechannelpotentiators.JPharmacolExpTher,330,783.
3. CateniF,ZacchignaM,PedemonteN,GaliettaLJ,MazzeiMT,FossaP,GiampieriM,MazzeiM.(2009)Synthesisof4-thiophen-2"-yl-1,4-dihydropyridinesaspotentiatorsoftheCFTRchloridechannel.BioorgMedChem,17,7894.
4. ChalothornD,ZhangH,SmithJE,EdwardsJC,FaberJE.(2009)Chlorideintracellularchannel-4isadeterminantofnativecollateralformationinskeletalmuscleandbrain.CircRes,105,89.
5. GoodchildSC,HowellMW,CordinaNM,LittlerDR,BreitSN,CurmiPM,BrownLJ.(2009)OxidationpromotesinsertionoftheCLIC1chlorideintracellularchannelintothemembrane.EurBiophysJ,39,129.
6. LiY,O"NeillC,DayML.(2009)Activationofachloridechannelbyatrophicligandisrequiredfordevelopmentofthemousepreimplantationembryoinvitro.BiolReprod,81,759.
7. MaH,JoYJ,MaY,HongJT,KwonBM,OhKW.(2009)ObovatolisolatedfromMagnoliaobovataenhancespentobarbital-inducedsleepingtime:PossIBLeinvolvementofGABAAreceptors/chloridechannelactivation.Phytomedicine,16,308.
8. McCaveraS,RogersAT,YatesDM,WoodsDJ,WolstenholmeAJ.(2009)Anivermectin-sensitiveglutamate-gatedchloridechannelfromtheparasiticnematodeHaemonchuscontortus.MolPharmacol,75,1347.
9. PiernoS,CamerinoGM,CipponeV,RollandJF,DesaphyJF,DeLucaA,LiantonioA,BiancoG,KunicJD,GeorgeAL,Jr.,ConteCamerinoD.(2009)StatinsandfenofibrateaffectskeletalmusclechlorideconductanceinratsbydifferentlyimpairingClC-1channelregulationandexpression.BrJPharmacol,156,1206.
10. PirriJK,McPhersonAD,DonnellyJL,FrancisMM,AlkemaMJ.(2009)Atyramine-gatedchloridechannelcoordinatesdistinctmotorprogramsofaCaenorhaBDitiselegansescaperesponse.Neuron,62,526.
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 |
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