细胞传代培养与MTT法检测化学药品对体外培养细胞增殖及存活的影响

CellularsubcultureandtheeffectofMTTassayofscanningofperoxidetothegrowthandsurvivalofcellsinvivo.HuBoqiang1(20092293)DongZhiWei11(20092283)1SchoolofLifeScience,BeijingInstituteofTechnology,Beijing100081,ChinaCellularsubcultureassaysistherudimentalmethodofallcellularexperiments.Viasubculture,cellscouldsurvive,duplicateandpreservetheirowngenes.Inourexperiment,wetransferredthesubculturecellsto96-wellplate,treatingwithH2O2indifferentconcentrationsanddyinglivingcells(6251cellline)withMTA.Finally,wegottheeffectofH2O2indifferentconcentrationstothesurvivingandgrowthof6251cells.1.IntroductionHydrogenperoxide(H2O2)isthesimplestperoxide(acompoundwithanoxygen-oxygensinglebond).Itcangenerateinsidethecell.Furthermore,itisalsoastrongoxidizer(AccordingtoWikipedia,itemHydrogenperoxide)whichcanfataltothesurvivalofEukaryoticcells.However,thankstoCatalase(CAT),theenzymewhichcouldreduceH2O2,cellscouldsurvivalinvivo.AlthoughCATcouldreducethesideeffectofH2O2,excessiveconcentrationofH2O2cancauseseriousdiseasestopeople,especiallyinagedgroupswhoseCATactivitywerelowerthantheyoungones.Paradoxically,H2O2playsa“double-edgeblade”roleinthepathologyofcancer.Ontheonehand,itcanintervenecellularDNArepairment,intracellularsignalstransferpathway,theadherenceofthecellandpromotingtheexpressionofVEGF,whichcouldacceleratethespreadoftumor.Ontheotherhand,however,H2O2,theindicatorofOxidativeStress,caninducetocellapoptosisanddepresstheduplicationofcells.SodoctorsusuallytakeadvantageofH2O2,likeRadiotherapy(usingUltraviolenttoproduceH2O2andotherperoxide)totrytocurecancer(Yao&Zhong,2006).Peroxidecanbefoundeverywhereindailylife.Forexample,inhairdye,peroxidecanmakehairmoreflexiblebycuttingupthedisulfidebondsbetweendifferentkeratinchains.Aswedonotknowwhetherperoxidewouldbeharmfuland,ifnotharmful,whatisthemaximumconcentrationofperoxideforhumancells,anexperimentisrequiredtoexaminethat.MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide,ayellowtetrazole),isreducedtopurpleformazaninlivingcells.Asolubilizationsolutionisaddedtodissolvetheinsolublepurpleformazanproductintoacoloredsolution.Theabsorbanceofthiscoloredsolutioncanbequantifiedbymeasuringatacertainwavelength(usuallybetween500and600nm)byaspectrophotometer,andbecausetheabsorbanceshowssignificantrelevanttothenumberoflivingcells,itisagoodwaytocountthenumberofthem.Amainapplicationallowsassessingtheviability(cellcounting)andtheproliferationofcells(cellcultureassays).Itcanalsobeusedtodeterminecytotoxicityofpotentialmedicinalagentsandtoxicmaterials,sincethoseagentswouldstimulateorinhibitcellviabilityandgrowth.Thisassaywouldbeusedinthisexperimenttodetecttheeffectofperoxideto6251cells.(AccordingtoWikipedia,item“MTTAssay”)2TheprocessoftheexperimentInstrument:Microplatereader,invertedmicroscope,counteringplate,96-wellplate,Pipette(Eppendorf).Material:6251celllinesinvitro.Reagent:2mg/mlMTT,trypsin,PBSbuffer,FCS-PBS,DMSO.Steps:1.Inoculationofcell.Taketheplasticcultureplatefrominsulationcantotheaircleanbench.WipetheplatewithEthanol.Afterallethanolevaporate,settheplateonthealcoholburnertokillbacteriaonthesurfaceoftheplate.Opentheplate,thenremoveallnutrientfluidofftheplateandadd1mltrypsinintheplate.After5minute,ifallcellsbecomeroundandflowwiththeliquid(seeinthemicroscope),removealltrypsinsolutionandadd2mlnutrientfluid.Flushthebottomoftheplateforatleast5mins,tomakesureallcellsaremovable.Thensuckasingledropoftheliquidtocountthenumberofcells.Addcellularsolutionsandbuffertothe96-wellplate(asdescribedinthetablebelow),thenculturethecellsfor26hoursin37℃tomakethecellspasteonthewallofthewells.H1GFEDCBA2150ulnutrientfluid150ulnutrientfluid150ulnutrientfluid3150ulcellularsolution150ulcellularsolution150ulcellularsolution4150ulcellularsolution150ulcellularsolution150ulcellularsolution5150ulcellularsolution150ulcellularsolution150ulcellularsolution6100ul100ul100ulcellularsolutioncellularsolutioncellularsolution7891011122.Addingdrugs:removingallliquidinrow3-6oflineGEF.Add100ul100umol/mlH2O2inrow3,200umol/mlH2O2inrow4and500umol/mlH2O2inrow5(Wefirstadded150ulinallwells,andafter30minallH2O2wereremovedandadded100ulH2O2solutioninstead,whichmaybethemostimportantreasonforthehighdeathrateinourfinallyresult).3.Washingtheplatewith200ul3%FCS-PBSforeachwell.BecarefultoinclinetheplatetomakeiteasytosucktheliquidandDONOTletthepipetteprickthebottomofthewell.4.SoluteallcellswithDMSO,200ulforeachwell.Keepshakingtheplatefor30minandthesolutionintheplatewouldturnpurple.5.PuttheplateintotheMicroplatereaderandreadOD550.3.Result3.1TheNumberoflivingcell5344Table1Numberforlivingcells=（𝐥𝐢𝐯𝐢𝐧𝐠𝐜𝐞𝐥𝐥𝐬𝐢𝐧𝟒𝐜𝐨𝐫𝐧𝐞𝐫𝐬）𝟒∗𝟏𝟎𝟒=𝟒×𝟏𝟎𝟒/mlAsthenumberoflivingcellsissuitableforfollowingsteps,wedidnotdilutethesolution.3.2SurvivalrateaftertreatedwithperoxideSurvivalrate=（𝐑𝐨𝐰𝐀𝐯𝐞𝐫𝐚𝐠𝐞−𝐄𝐦𝐩𝐭𝐲𝐂𝐨𝐧𝐭𝐫𝐨𝐥）（𝐂𝐞𝐥𝐥𝐜𝐨𝐧𝐭𝐫𝐨𝐥−𝐄𝐦𝐩𝐭𝐲𝐂𝐨𝐧𝐭𝐫𝐨𝐥）EmptyControl100umol/mlH2O2200umol/mlH2O2500umol/mlH2O2CellcontrolE0.0730.0820.0660.0710.288F0.0690.0720.0610.0680.668G0.0860.1140.0790.1010.427Average0.0760.0890.0690.0800.692*Surviva