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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Cell Meter™ Fluorimetric Phagocytosis Assay Kit *Red Fluorescence*/21225/100 Tests
产品编号:21225
市  场 价:¥85560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
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美  元  价:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Cell Meter™ Fluorimetric Phagocytosis Assay Kit *Red Fluorescence*/21225/100 Tests
商品介绍
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Ex/Em(nm)575/597
MWN/A
CAS#N/A
SolventN/A
StorageR/L
CategoryCellBIOLOGy
pHandIonIndicators
RelatedCellMetabolism
Phagocytosisisdefinedasthecellularuptakeofparticleswithinaplasma-membraneenvelopebyacell.TheprocessofphagocytosisiscriticalintheinnateimmuneresponsebyengulfmentanddestructionofinvADIngmicroorganisms.Phagocytosisisalsorequiredinmaintainingtissuehomeostasisandremodelingbytheclearanceofapoptoticbodies.Theuptakemechanismofphagocytosisdependsonthesizeoftheparticles,receptor-ligandinteractions,andinvolvementoftheCytoskeleton.Onceinternalized,thephagosomefuseswithlysosomestoformsecondaryphagolysosomefordigestion,resultinginprogressivedecreaseofpH.OurCellMeter™FluorimetricPhagocytosisAssayKitutilizesauniquepHdependentProtonex™600Red-latexbeadconjugates.ThebeadsareinreadytousesUSPension.Unlikemostoftheexistingfluorescentdyes,theProtonex™600Red-latexbeadconjugateisnon-fluorescentoutsideofthecells.However,itsfluorescencedramaticallyincreasesastheyareinsidetheacidicphagosomes/phagolysosomes.Thischaracteristicmakesiteasytousewithouttrypanbluequenchingstepandarobusttooltostudyphagocytosisanditsregulations.CellMeter™FluorimetricPhagocytosisAssayKitalsoincludesagreenfluorescentcellviABIlitydye,whichallowingthesimultaneousdetectionofbothlivecellsandtheprocessofphagocytosisbyfluorescentmicroscopy.Thisassaycanalsobeadaptedforfluorescencemicro-platereaderandflowcytometrydetections.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
  1. Preparecells:
    1. Foradherentcells:Platecellsovernightingrowthmediumat20,000-50,000cells/well/100µLfora96-wellplate.
      Note1:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensity.
      Note2:ForRAW264.7cellsthatweusedinthisassaywerecommendtoplatecellsat50,000cells/well/100µLin96-wellplateforovernight.
  2. Preparestocksolutions:
    1. Prepare6XCytochalasinD:Add18µLof10mMCytochalasinD(finalconcentrationinthewellis10µM)or18µLofDMSOin3mLcellgrowthmedium.
    2. Prepare12XProtonex™600Red-LatexBeadsConjugatesolution:Add8µLofProtonex™600Red-LatexBeadsConjugate(ComponentA)in2mLcellgrowthmedium(containing10%FBS).
      Note:Theunusedbeadscanbestoredat4°C.
    3. Prepare12XCytoTrace™Green:Add20µLofDMSO(ComponentC)intothevialofCytoTrace™Green(ComponentB)tomake400Xstocksolution.Andthenadd5µLin2mLcellgrowthmediumtohave12XCytoTrace™Greensolution
      Note:TheunusedCytoTrace™GreenDMSOstocksolutioncanbealiquotedintosingleusevialsandstoredat-20°C.
  3. Phagocytosisassayprotocol:
    1. Add25µLof6XCytochalasinD(fromstep2.1)orDMSOsolution(fromstep2.1)intoeachwell.
      Note:TheconcentrationofCytochalasinDusedintheassayshouldbeoptimizedforeachindividualcellline.
    2. Incubatetheplateinthecellincubatorfor30minutes.
    3. Add12.5µLof12XProtonex™600Red-LatexBeadsConjugatesolution(fromstep2.2)intoeachwell.
    4. Incubatetheplateinthecellincubatorfor4hours.
      Note:Theincubationtimeshouldbeoptimizedbyusersforeachindividualcelllines.
    5. Add12.5µLof12XCellTracker(fromstep2.3)intoeachwell.
    6. Incubatetheplateinthecellincubatorfor30minutes.
    7. Washtheplatetwicewith1XPBS.
  4. ObservephagocytosisinsidethecellswithTexasRedfilter(Ex/Em=570/600nm)andCytoTrace™GreenwithFITCfilter(Ex/Em=490/525nm)
References&Citations
CitationExplorer

PHD2isaregulatorforglycolyticreprogramminginmacrophages
Authors:AnnemarieGuentsch,AngelikaBeneke,LijaSwain,KatjaFarhat,ShunmugamNagarajan,BenWielockx,KaaminiRaithatha,JanDudek,PeterRehling,AnkeZieseniss
Journal:MolecularandCellularBiology(2016):MCB--00236


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