流式细胞术检测细胞存活率的方法学建立

*刘阳1,赖翼1,李敏惠1,杨淑霞1,郑静1,阳泰1,李丽梅1,邓勇1,张强1,任荣梅1,李钱梅1,张帅1,邹强1,李华2(1.成都医学院科研中心610083;2.中国人民解放军成都军区总医院肿瘤科,成都610083):探讨流式细胞术测定细胞存活率的可行性及准确性,为药物筛选细胞增殖测定药物毒性检测等提供新的选择使用流式细胞术CCK-8比色法以及台盼蓝染色排除法测定Jurkat细胞存活率使用流式细胞术,细胞浓度大于105/mL时,CV值小于5%;细胞浓度小于104/mL时,CV值为5%~10%使用流式细胞术r2为0.9935,斜率为0.9994;使用CCK-8比色法r2为0.9912,斜率为1.011;使用台盼蓝染色排除法r2为0.9698,斜率为1.057流式细胞术进样流速稳定,且与活细胞数量成正比,可用于测定细胞存活率:流式细胞术;辛卡利特;台盼蓝染色排除法;细胞流速DOI:10.3969/j.issn.1673-4130.2011.15.003:A:1673-4130(2011)15-1663-02Developmentofanassayforthedetectionofthesurvivalratesofcellsbyflowcytometry*LiuYang1,LaiYi1,LiMinhui1,YangShuxia1,ZhengJing1,YangTai1,LiLimei1,DengYong1,ZhangQiang1,RenRongmei1,LiQianmei1,ZhangShuai1,ZouQiang1,LiHua2(1.ResearchCenter,ChengduMedicalCollege,Chengdu610083,China;2.DepartmentofOncology,ChengduMilitaryGeneralHospital,Chengdu610083,China)Abstract:ObjectiveToinvestigatethefeasibilityandaccuracyofflowcytometry(FCM)forthedetectionofthesurvivalratesofcellstoprovideanotherchoiceofdrugscreen,cellproliferationassayanddrugtoxicityassay.MethodsFlowcytometry,CCK-8andtrypanbluestainingtechniquewasperformedforthedetectionofthesurvivalratesofJurkatcells.ResultsThecoefficientofvariation(CV)ofFCMwaslessthan5%,whenthecellconcentrationexceed105/mLandwas5%-10%,whenthecellconcentra-tionwasbelow105/mL.Ther2andslopeofFCMwere0.9935and0.9994respectively,thoseofCCK-8were0.9912and1.011respectivelyandthoseoftrypanbluestainingtechniquewere0.9698and1.057respectively.ConclusionTheflowrateofsampleintroductionofFCMcouldbestableandwithdirectproportionwiththeamountofviablecells.FCMcouldbeusedforthedetectionofthesurvivalrateofcells.Keywords:flowcytometry;sincalide;trypanbluetechnique;celldetectionspeed,[1][2][3][4]MTT[5]CCK-8(cellcountingkit-8)[6][7],,,,,,,[8],,,,,,(PI)7-DeFluor660,,,,TJurkat,CCK-8,,11.1TJurkat(ATCC)1.2BDFACSCaliburBioteckPowerwaveTS100RPMI-1640GIBCOCCK-8DojindoPISigma1.31.3.1Jurkat10%RPMI-164037,5%CO2106/mL105/mL104/mL103/mL5g/mLPI10min,PI1663国际检验医学杂志2011年9月第32卷第15期IntJLabMed,September2011,Vol.32,No.15*:(No.81072455);(No.209104);(NCET-09-0888),E-mail:[e-mail]1.3.2CCK-8JurkatJurkat1.25106/mL,200L,250103100%,90%80%70%60%50%40%30%20%10%5%1%,22510320010317510315010312510310010375103501032510312.51032.51035g/mLPI10min,PIJurkat;96,200L,CCK-83h,450nm(OD);,1.4SPSS14.0xs(CV),(r2)22.144,CV10%,105/mL,CV5%,2.2JurkatFSC-H,PI,Jurkat,,,12,r20.9935,,,0.99942.3CCK-8Jurkat,12OD(450nm),r20.9912,OD,1.0112.4Jurkat,12,r20.9698,,1.0572.533Jurkat,CV(5.3551.275)%,r20.99230.0012,0.99810.0018;CCK-8CV(5.4661.741)%,r20.99080.0007,1.0090.004;CV(12.7505.246)%,r20.96460.0046,1.0600.005,CCK-823CCK-82MTT[9],CCK-8MTT,MTT臜不溶于水的缺点,具有更高的检测[10],,,100,[7],,,,,,[1]LiQ,MaddoxC,RasmussenL,etal.Assaydevelopmentandhigh-throughputantiviraldrugscreeningagainstBluetonguevirus[J].AntiviralRes,2009,83(3):267-273.[2]YeYW,WuJH,WangCM,etal.Sox17regulatesproliferationandcellcycleduringgastriccancerprogression[J].CancerLett,2011,307(2):124-131.[3]YanF,ZhangC,ZhengY,etal.Theeffectofpoloxamer188onnanoparticlemorphology,size,cancercelluptake,andcytotoxicity[J].Nanomedicine,2010,6(1):170-178.[4]WangCX,HuangLS,HouLB,etal.Antitumoreffectsofpolysor-bate-80coatedgemcitabinepolybutylcyanoacrylatenanoparticlesinvitroanditspharmacodynamicsinvivoonC6gliomacellsofabraintumormodel[J].BrainRes,2009,1261:91-99.[5]FischerD,LiY,AhlemeyerB,etal.Invitrocytotoxicitytestingofpolycations:influenceofpolymerstructureoncellviabilityandhemolysis[J].Biomaterials,2003,24(7):1121-1131.[6]ChenCL,LinCF,ChangWT,etal.Ceramideinducesp38MAPKandJNKactivationthroughamechanisminvolvingathioredoxin-interactingprotein-mediatedpathway[J].Blood,2008,111(8):4365-4374.[7]LouisKS,SiegelAC.Cellviabilityanalysisusingtrypanblue:manualandautomatedmethods[J].MethodsMol,2011,740:7-12.[8]OkadaS,MakinoH,NagumoA,etal.CirculatingCD34-positivecellnumberisassociatedwithbrainnatriureticpeptidelevelintype2diabeticpatients[J].Diabetescare,2008,31(1):157-158.[9]MosmannT.Rapidcolorimetricassayforcellulargrowthandsur-vival:applicationtoproliferationandcytotoxicityassays[J].JIm-munolMethods,1983,65(1/2):55-63.[10]TominagaH,IshiyamaM,OhsetoF,etal.Awater-solubletet-razoliumsaltusefulforcolorimetriccellviabilityassay[J].AnalCommun,1999,36(2):47-50.(:2011-06-10)1664国际检验医学杂志2011年9月第32卷第15期IntJLabMed,September2011,Vol.32,No.15