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AntibodiesOnline/96Well Cellular Senescence Assay Kit (SAβgal Activity, Fluorometric Format)/ABI188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/96Well Cellular Senescence Assay Kit (SAβgal Activity, Fluorometric Format)/ABI

Reactivity
Mammalian
Application
CellularAssay(CA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
SampleTypeCellSamples
DetectionMethodFluorometric
Components
  1. 2XCellLysisBuffer:One2mLtube
  2. 2XReactionBuffer:One2mLtube
  3. SA-ß-GalSubstrate(20X):One75μLambertube
  4. StopSolution:Three2mLtubes2
Materialnotincluded
  1. Senescentcellsortissuesamples
  2. 37°CIncubator
  3. β-mercaptoethanol
  4. 96-wellplatesuitableforafluorescenceplatereader
  5. 96-wellFluorometer
  6. ProteinAssayReagents
BackgroundNormalprimarycellsproliferateincultureforalimitednumberofpopulationdoublingspriortoundergoingterminalgrowtharrestandacquiringasenescentphenotype.Thisfinitelifespancorrelateswiththeageoftheorganismandwiththelifeexpectancyofthespeciesfromwhichthecellswereobtained,suchthattheoldertheageortheshorterthelifespan,thelesstheabilityofthecellstoundergopopulationdoubling.SenescentcellsarecharacterizedbyanirreversibleG1growtharrestinvolvingtherepressionofgenesthatdrivecellcycleprogressionandtheupregulationofcellcycleINK4aCIP1inhibitorslikep16,p53,anditstranscriptionaltarget,p21.Theyareresistanttomitogen-inducedproliferation,andassumeacharacteristicenlarged,flattenedmorphology.Researchintothepathwaysthatpositivelyregulatesenescenceandwayscellsbypasssenescenceisthereforecriticalinunderstandingcarcinogenesis.Normalcellshaveseveralmechanismsinplacetoprotectagainstuncontrolledproliferationandtumorigenesis.SenescentcellsshowcommonbiochemicalMarkerssuchasexpressionofanacidicsenescence-associatedß-galactosidase(SA-ß-Gal)activity.Whilesenescencehasbeencharacterizedprimarilyinculturedcells,thereisalsoevidencethatitoccursinvivo.CellsexpressingmarkersofsenescencesuchasSA-ß-Galhavebeenidentifiedinnormaltissues.The96-wellCellularSenescenceAssayKitprovidesaneasy-to-useandefficientmethodtodeterminethecellularsenescencebymeasuringSA-ß-Galactivityusingafluorometricsubstrate.ThisquantitativeassayusescelllysateforbothSA-β-galactosidaseactivitydeterminationandnormalizationofsamplescontainingdifferentcellnumbers.EachTrialSizekitprovidessufficientquantitiestoperformupto24assaysina96-wellplate.
ApplicationNotesOptimalworkingdilutionshouldbedeterminedbytheinvestigator.
Comment

  • Measureactivityofsenescence-associatedß-galactosidase
  • Quantitativeresultsinafluorescenceplatereader

ReagentPreparation

1XCellLysisBuffer:Preparea1XCellLysisBufferbydilutingtheprovided2Xstock1:2inddH2O.Storethedilutedsolutionatroomtemperatureforuptosixmonths.Immediatelybeforeuse,addproperamountofproteinaseinhibitorssuchasPMSF.2XAssayBuffer:Immediatelybeforeuse,addβ-mercaptoethanolto2XReactionBufferatafinalconcentrationof10mManddilute20XSA-ß-GalSubstrateto1Xwith2XReactionBuffercontaining10mMβ-mercaptoethanol

  • Dontstore2XAssayBuffer.Reagent96-well24-well6-well10cm1XCellLysisBuffer100μL400μL1000μL1500μLTable1.1XCellLysisBufferRequiredperWellforVariousCulturePlates.

AssayProcedure
  1. Aspiratethemediumfromthesenescentcells.
  2. Washthecellsoncewith200μLofcold1XPBSandaspiratethewash.
  3. Add100μLofcold1XCellLysisBuffer(seethetableabovefortherequiredamountof1XCellLysisBufferofotherplateformats).Incubateat4 °Cfor5 minutes.Transferthewholelysatetoamicrocentrifugetubeandcentrifuge10 minutesat4 °C.Collectsupernatantascelllysate.
  4. (optional)DeterminethetotalproteinconcentrationofeachcelllysatesamplebyproteinassaysuchasPiercesBCAproteinAssay.
  5. Transfer50μLofthecelllysatetoa96-wellplate.Add50μLoffreshlyprepared2XAssayBuffer.Incubatethewellsat37 °Cprotectedfromlightfor1-3hr.
  6. Remove50μLofthereactionmixturetoa96-wellplatesuitableforfluorescencemeasurement.Stopthereactionbyadding200μLofStopsolution.
  7. Readfluorescencewithafluorescenceplatereaderat360nm(Excitation)/465nm(Emission).3
RestrictionsForResearchUseonly
Storage4°C/-20°C
StorageCommentStoreSA-β-galsubstratesolutionprotectedfromlightat-20°C.Storeallothercomponentsatroomtemperature.
SupplierImages
Cellular Assay (CA) image for 96-Well Cellular Senescence Assay Kit (SA-β-gal Activity, Fluorometric Format) (ABIN2344914)SA-ß-GalactivityinSenescentHumanLungFibroblastHFL-1Cells. NormalHFL-1cells...
Productcitedin:Hu,Sung,Jessen,Thibault,Finkelstein,Khan,Sacaan:"MechanisticInvestigationofBoneMarrowSuppressionAssociatedwithPalbociclibanditsDifferentiationfromCytotoxicChemotherapies."in:Clinicalcancerresearch:anofficialjournaloftheAmericanAssociationforCancerResearch,2016(PubMed).

Kim,Lee,Ko,Chun,Kim,Sung,Koo,Yoo:"Cellculturedensityaffectstheproliferationactivityofhumanadiposetissuestemcells."in:Cellbiochemistryandfunction,Vol.34,Issue1,pp.16-24,2016(PubMed).

Platas,Guillén,Gomar,Castejón,Esbrit,Alcaraz:"Anti-senescenceandAnti-inflammatoryEffectsoftheC-terminalMoietyofPTHrPPeptidesinOAOsteoblasts."in:Thejournalsofgerontology.SeriesA,Biologicalsciencesandmedicalsciences,Vol.72,Issue5,pp.624-631,2016(PubMed).

Zhang,Gong,Wang,Chen,Lim,Dolata,Chen:"Cryptosporidiumparvuminfectionattenuatestheex vivopropagationofmurineintestinalenteroids."in:Physiologicalreports,Vol.4,Issue24,2016(PubMed).

Shimizu,Kanno,Sugiyama,Ohata,Araki,Kishikawa,Kimura,Yamamoto,Kodama,Kihira,Tazuma:"Cholangiocytesenescencecausedbylysophosphatidylcholineasapotentialimplicationincarcinogenesis."in:Journalofhepato-biliary-pancreaticsciences,Vol.22,Issue9,pp.675-82,2015(PubMed).

Liao,Fang,Chai,Wu,Ho,Yang,Tzeng:"DepletionofBcellCLL/Lymphoma11BGeneExpressionRepressesGliomaCellGrowth."in:Molecularneurobiology,2015(PubMed).

Hirano,Murakami,Ono,Sakurai,Tominaga,Takahashi,Nagai,Doi,Abe:"ANovelInteractionbetweenFLICE-AssociatedHugeProtein(FLASH)andE2ARegulatesCellProliferationandCellularSenescenceviaTumorNecrosisFactor(TNF)-Alpha-p21WAF1/CIP1Axis."in:PLoSONE,Vol.10,Issue7,pp.e0133205,2015(PubMed).

Chang,Huo,Li,Wu,Zhang:"Ascorbicacidprovidesprotectionforhumanchondrocytesagainstoxidativestress."in:Molecularmedicinereports,Vol.12,Issue5,pp.7086-92,2015(PubMed).

Lu,Zhang,Chen,Lu,Yang,Liu,Ma:"SIRT1counteractedtheactivationofSTAT3andNF-κBtorepressthegastriccancergrowth."in:Internationaljournalofclinicalandexperimentalmedicine,Vol.7,Issue12,pp.5050-8,2015(PubMed).

Li,Liu,Zhou,Kelley,Edwards,Gao,Qiao:"InhibitionofAPE1/Ref-1redoxactivityrescueshumanretinalpigmentepithelialcellsfromoxidativestressandreduceschoroidalneovascularization."in:Redoxbiology,Vol.2,pp.485-94,2014(PubMed).

Klinkhammer,Kramann,Mallau,Makowska,vanRoeyen,Rong,Buecher,Boor,Kovacova,Zok,Denecke,Stuettgen,Otten,Floege,Kunter:"Mesenchymalstemcellsfromratswithchronickidneydiseaseexhibitprematuresenescenceandlossofregenerativepotential."in:PLoSONE,Vol.9,Issue3,pp.e92115,2014(PubMed).

Qi,Zuo,Bai,Xu,Li,Shen,Wang,Zhang,Wu:"COH-203,anovelmicrotubuleinhibitor,exhibitspotentanti-tumoractivityviap53-dependentsenescenceinhepatocellularcarcinoma."in:Biochemicalandbiophysicalresearchcommunications,Vol.455,Issue3-4,pp.262-8,2014(PubMed).

Malhotra,Portales-Casamar,Singh,Srivastava,Arenillas,Happel,Shyr,Wakabayashi,Kensler,Wasserman,Biswal:"GlobalmappingofbindingsitesforNrf2identifiesnoveltargetsincellsurvivalresponsethroughChIP-Seqprofilingandnetworkanalysis."in:Nucleicacidsresearch,Vol.38,Issue17,pp.5718-34,2010(PubMed).


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