Brdu 细胞增殖检测实验.docx

Brdu 细胞增殖检测实验.docx

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Brdu细胞增殖检测实验

Brdu检测细胞增殖试验

试验操纵:

1．铺细胞,每个3.5cmdish10万个,在37℃.5%CO2孵箱中造就72h（细胞密度至50-60%阁下）.

2．Brdu（5-溴-2′-脱氧尿苷）参加造就细胞中,1mg/ml,标识表记标帜48h.（量：

Brdu以铺满全部dish底面为准.）

3．固定：

PBS洗细胞爬片3次,每次5min,在摇床上晃悠清洗,4%PFA固定30min.

4．变性：

将固定好的细胞爬片用PBS洗3次,每次5min,2mol/L的HCl在37℃前提下变性5min,可放置于37℃恒温孵箱,运用封口膜把造就皿封好.（120r/m）

5．中和：

0.1mol/L的硼酸钠（PH8.3）中和10min,PBS洗3次,每次5min.（50r/m）

6．参加1ml的0.2%TritonX-100,10min.

7．吸出TritonX-100,用PBS洗3次,每次5min.

8．参加1ml3%的BSA关闭,室温1h,可在摇床上晃悠.

9．吸出BSA,用PBS洗3次,每次5min.

10．加一抗（尿嘧啶脱氧核苷Brdu（鼠单抗）1:

200）,用1%BSA稀释,4渡留宿.

11．将孵好一抗的细胞爬片用PBS洗3次,每次10min.

12．加二抗（羊抗鼠IgG/AlexaFluor5941:

100）,用1%BSA稀释,避光室温孵育1h.（60r/min）

13．将孵好二抗的细胞爬片用PBS洗3次,每次10min.

14．加DAPI染细胞核,储存浓度为1mg/ml,应将DAPI完整混匀,可用手弹几下,一般稀释比例为1:

1000（用PBS稀释）,避光室温反响10min.

15．将DAPI染好的细胞爬片用PBS洗3次,每次10min.

16．中性树胶封片,荧鲜明微镜不雅察,200×镜下取5个视野,计数Brdu阳性细胞和蓝染的细胞核数量,然落后行统计剖析.

试剂配制：

a.Brdu的消融：

室温下,将250mg粉末溶于2.5ml的DMSO中,储存浓度为100mg/ml分装,每管120ul,-20℃保管.

b.2mol/LHCl：

取8.333mL12mol/LHCl的浓HCl,参加DDW定容至50mL.

c.0.1mol/L硼酸钠：

称量1.907g硼砂（Na2B4·10H2O381.36g/mol）,参加DDW定容至50mL,调PH=8.3.

d.0.2%TritonX-100：

有0.5%的Triton-100（2.5mL原液消融于47.5mL的PBS中）,用PBS稀释至0.2%.

e.3%BSA：

称量1.5gBSA,消融于50mL的PBS中.

f.1%BSA：

用PBS稀释3%BSA至1%.

g.4%FPS：

4%多聚甲醛.

我是用DDW配制的,后来我发明很难溶,磁力搅拌器加热搅拌,固然温度掌握在60℃以下,也老是放心多聚甲醛分化为甲醛,所以,我就总结为如下：

提前配制.

4%多聚甲醛溶液（pH7.2）试剂：

多聚甲醛（PFA）4gDDW　至100ml

配制办法：

称取4g多聚甲醛（粉末状）,置于三角烧瓶中,参加80mlDDW,放入37恒温水浴箱,每隔1-2小时摇摆混匀,16-24小时PFA会完整消融.填补DDW,调节PH值.

试验道理：

1.免疫染色试验的基起源基础理

运用固定剂（平日是甲醛或多聚甲醛）将细胞固定,使得细胞膜的通透性大大增长,并且运用Triton-X-100使得一部分膜蛋白变性,从而使通透性进一步加强.运用正常羊血清关闭,可以令很多蛋白先与血清内的非特异性抗体联合,而特异性的抗体因为动力学的关系可以经由过程竞争性的反响与目标蛋白联合,这一进程可以包管抗体识此外特异性.二抗可以特异性辨认一抗的Fc区域,运用二抗衔接不合的荧光基团,就可以在荧鲜明微镜下不雅察到不合的荧光,从而显示目标基因的表达情形.

2.BrdU标识表记标帜道理

细胞增殖周期包含G1.S.G2.M4个时代,个中S期是DNA合成期,细胞内DNA进行半保存复制,各类组成DNA的原料掺入到DNA中.BrdU作为一种胸腺嘧啶核苷的相似物（其化学构造特色是胸腺嘧啶的碱基嘧啶环上与5位C原子衔接的甲基被溴代替）,像胸腺嘧啶核苷一样可掺入到细胞合成的DNA中.当细胞处于DNA合成期（S期）而同时又有BrdU消失时,就会有BrdU掺入新合成的DNA中,只要细胞不必亡,这种BrdU就在胞核的DNA中长期存留.掺入到DNA的BrdU可经由过程抗BrdU单克隆抗体在组织切片或细胞爬片上显示.

BrdU抗体比较大,因为DNA双链构造的位阻,BrdU抗体无法直接与双链上的BrdU联合,必须先用使DNA部分变性,如许变性了的DNA单链上的BrdU才干与BrdU抗体联合,是以做BrdU细胞增殖试验必定要变性,当然变性的办法包含酸解,热解等,但是要留意变性的程度也很主要.建议采取EdU细胞增殖检测办法,无需变性,无需酶解,无需抗体,小分子染色,3小时完成试验.EdU是一种胸腺嘧啶核苷相似物,可以或许在细胞增殖时代代替T渗入正在复制的DNA分子,经由过程基于EdU与Apollo?

荧光染料的特异性反响检测DNA复制活性,通快速.更敏锐.更精确.EdU检测染料只有BrdU抗体大小的1/500,在细胞内很轻易集中,无需DNA变性（酸解.热解.酶解等）即可有用检测,可有用防止样品毁伤,在细胞和组织程度能更精确地反应细胞增殖等现象.

该办法能对细胞周期进行敏捷而稳固的测量,并且标识表记标帜BrdU的细胞只要不受到紫外线照耀,对细胞本身没有功效伤害.该技巧可运用到跟踪检测移植细胞的存活.分化和功效状况.

3.DAPI染色道理

DAPI为4’,6二脒基-2-苯吲哚（4’,6—diamidino-2—phenylindole）,能与双链DNA小槽,特殊是AT碱基联合,也可拔出少于3个持续AT碱基对的DNA序列中.当它与双链DNA结应时,荧光强度加强20倍,而与单链DNA联合则无荧光加强现象,是以是一种简略单纯.快速和迟钝地检测DNA的办法.DAPI的荧光强度虽较Hoechst低,但荧光稳固性优于Hoechst;其特异性较溴化乙啶（ethidliumbromide,EB）和碘化丙啶（propidiumiodide,P1）高.DAPI的中文名称是4,6-联脒-2-苯基吲哚,是一种经常运用的荧光染料,其感化机理与溴化乙锭（EB）等染色剂的机理相似：

它们与DNA双螺旋的凹槽部分可以产生互相感化,从而与DNA的双链慎密联合.联合后产生的荧光基团的接收峰是358nm而散射峰是461nm,正好UV（紫外光）的激发波长是356nm,使得DAPI成为了一种经常运用的荧光检测旌旗灯号.

ANALYSISOFCELLCYCLE

1.INTRODUCTION

Cellcycleandapoptosisareveryimportantfunctionalparameterstoassessthecellularmetabolism,physiologyandpathology.Severaltechniqueshavebeendevelopedtoquantitatetheseparametersutilizingthedifferentialstainingoffluorescentdyes.Wearedescribingfourdifferentflowcytometricmethods,twoforthediscriminationofcellcyclephases（AandB）andtwoforthesimultaneousassessmentofcellcycleandapoptosis（CandD）.

A）Bromodeoxyuridine/PropidiumIodide

TheclassicalmethodfortheanalysisofcellcycledistributionistheflowcytometricmeasurementofDNAcontentwhichcansimultaneouslydeterminetheincorporationofBromodeoxyuridine（BrdU）.TheprocedurerequiresthatDNAispartiallydenaturedtoexposeincorporatedBrdUtoaspecificantibody.DenaturationisnecessarybecauseantibodiesdevelopedsofarbindonlytoBrdUinsingle-strandDNA.TheremainingundenaturedDNAisthenstainedwithPropidiumIodide（PI）.Greenfluorescencefromthefluorescein-conjugatedantibodyisameasureofBrdUincorporation.RedfluorescencefromthePIisameasureofDNA.Theprotocoldescribedhereuseshigh-molarityHClforthedenaturationofDNA.Furthermore,thismethodmaybeutilizedeitherforunfixedorforfixedcellsinsuspension.

B）Cyclins/PropidiumIodide

Cyclinsarekeycomponentsofthecellcycleprogressionmachinery.Inparticular,theexpressionofcyclinsD,E,AandB1providesnewcellcyclelandmarksthatcanbeusedtosubdividecellcycleintoseveraldistinctsubcompartments.Inthisprocedurecyclinsexpressionisdetectableusingspecificmonoclonalantibodies（mAbs）,andisanalysedinrespecttoDNAcontent.

Generally,thepeakofexpressionofcyclinD1canbedetectedinearlyG1,thepeakofcyclinEistypicalofG1/Stransition,thepeakofcyclinAcanbedetectedduringG2/MphasesandcyclinB1istypicaloflateG2/M.Usingthismethod,comparedtotheabovementionedprotocol,itispossibletodistinguishG0fromG1andG2fromMphases.However,itisnecessarytokeepinmindthatnotallcelltypesbehaveinthesamemanner（forexample,cyclinD1isdetectablenotonlyinG0/G1butalsoinG2/M,evenifinaveryfewcelltypes）.

C）TUNEL/PropidiumIodide

Oneofthemostusedprotocolforthedeterminationofapoptosisinthedifferentphasesofcellcycleistheenzymaticinsitulabelingofapoptosis-inducedDNAstrandbreaks（TUNEL）.Terminaldeoxynucleotidyltransferase（TdT）havebeenusedfortheincorporationoffluorescein-labelednucleotidestoDNAstrandsbreaksinsitu.DNAcontentisrevealedbyredfluorescencefromPI.Inordertohavemoredetails,seetheChaptersrelatedtoTUNELtechnique.

D）F-Actin/PropidiumIodide

Theanalysisofapoptoticcellsandestimationoftheircellcyclespecificityisalsopossibleusingarecentmethod.ThisisbasedonidentificationofapoptoticcellswhichhavemodifiedtheircytoskletonandtheirDNAcontent.Inspecific,paraformaldehyde（PFA）fixationfollowedbystainingofF-actinwithfluorescein-conjugatedphalloidinandofDNAwithPI,areused.Furthermore,thisproceduremaybeutilizedalsoforadherentcells.

A）BrdU/PIPROTOCOL

A.2.1Materials

BrdU（A2）,washingbuffer（A1）,HCl4M,Boraxbuffer（A1）,anti-BrdUantibody（A2）,goat-anti-mouse-FITCantibody（A2）,PIbuffer（A1）.A.2.2.Methodology

1.Cells（1x106/mL）areincubatedwithBrdU10mMatfinalconcentration,for30minat37°Cincontrolledatmosphere.

2.Washtwiceat500gfor1minusingthewashingbuffer.

3.Resuspendin0.5mLofwashingbufferand0.5mLofHCl4M.

4.Mixaccuratelyandincubatefor30minatroomtemperature.

5.Washonceasinstep2.

6.Resuspendin1mLofBoraxbuffer.

7.Asinstep5.

8.Resuspendin200mLofwashingbufferandlabelwith5mLofmAbant-BrdU.

9.Incubatefor1hourat4°Cinthedark.

10.Asinstep5.

11.Resuspendin200mLofwashingbufferandlabelwith4mLofgoat-anti-mouseFITC-conjugatedantibody.

12.Incubatefor30minat4°Cinthedark.

13.Asinstep5.

14.Resuspendin200mLofwashingbufferand200mLofPIbuffer.

15.Incubatefor15-30minat4°Cinthedark.

16.Analysewithflowcytometerequippedwitha488nmargonlaser.

A.3.COMMENTARY

A.3.1Backgroundinformation

Inthisprocedurefixedcellsby4%PFAinPhosphateBufferSaline（PBS）canbeutilized.InthiscasetowashcellsonceinPBSbeforetostartatstep1isnecessary.

Moreover,bothdirectandindirectimmunofluorescencecanbeused.TheBrdUincorporationismoreevidentusingtheindirectmethod.

A.3.2Anticipatedresults

Itisrecommandedtoperformeachexperimentusinganegativeandapositivecontrolsample.Thenegativesample,inordertohaveacorrectsettingofinstrument,isassessedfollowingallthestepsexceptstep8.Thepositivesample,inordertomakesurethatthemethodworks,isassessedbyusingaproliferatingcellline（suchasU937,K562,MOLT4,etc.）followingallsteps.A.3.3Timeconsiderations

Theprotocolissimplybutitrequireaquitelongtime.Indeed,forfewsamples,moreorless4hoursarerequired.Thedurationofthemethodisobviouslydependingonthenumberofsamples.A.3.4Keyreferences

1.Dolbeare,F.,Gratzner,H:

Pallavicini,M.,Gray,J.W.1983.Proc.Natl.Accad.Sci.U.S.A.80:

5573.