JournalofVisualizedExperimentswww.jove.comCopyright©2011JournalofVisualizedExperimentsAugust2011|54|e2899|Page1of4VideoArticleInductionandTestingofHypoxiainCellCultureDanliWu1,PatriciaYotnda11CenterforCellandGeneTherapy,BaylorCollegeofMedicineCorrespondenceto:PatriciaYotndaatpyotnda@bcm.eduURL:http://www.jove.com/video/2899DOI:doi:10.3791/2899Keywords:CellBiology,Issue54,mammaliancell,hypoxia,anoxia,hypoxiainduciblefactor(HIF),reoxygenation,normoxiaDatePublished:8/12/2011Citation:Wu,D.,Yotnda,P.InductionandTestingofHypoxiainCellCulture.J.Vis.Exp.(54),e2899,doi:10.3791/2899(2011).AbstractHypoxiaisdefinedasthereductionorlackofoxygeninorgans,tissues,orcells.Thisdecreaseofoxygentensioncanbeduetoareducedsupplyinoxygen(causesincludeinsufficientbloodvesselnetwork,defectivebloodvessel,andanemia)ortoanincreasedconsumptionofoxygenrelativetothesupply(causedbyasuddenhighercellproliferationrate).Hypoxiacanbephysiologicorpathologicsuchasinsolidcancers1-3,rheumatoidarthritis,atherosclerosisetc…Eachtissuesandcellshaveadifferentabilitytoadapttothisnewcondition.Duringhypoxia,hypoxiainduciblefactoralpha(HIF)isstabilizedandregulatesvariousgenessuchasthoseinvolvedinangiogenesisortransportofoxygen4.Thestabilizationofthisproteinisahallmarkofhypoxia,thereforedetectingHIFisroutinelyusedtoscreenforhypoxia5-7.Inthisarticle,weproposetwosimplemethodstoinducehypoxiainmammaliancellculturesandsimpleteststoevaluatethehypoxicstatusofthesecells.VideoLinkThevideocomponentofthisarticlecanbefoundathttp://www.jove.com/video/2899/Protocol1.HypoxiainducedbyCoCl2solutionCobalt(II)Chloridehexahydrate(CoCl2•6H2O,MW=237.9)isachemicalinducerofhypoxia-induciblefactor-1.38.Thisproductissolubleinwater(100mg/ml),yieldingaclear,redsolution.1.Preparea25mMstocksolutioninsterileddwater,(prepareimmediatelybeforeuse)2.UseCoCl2atthefinalconcentrationof100μMinyourregularcellculturemediatoinducehypoxia.3.AddtheCoCl2containingmediatoyourcellsandincubatetheculturesfor24hoursinaconventionalincubator(37°C;5%C02).Theabov...
