请使用支持JavaScript的浏览器! 为什么大多聚合物溶解比较慢 说明为什么大多数聚合物的溶解缓慢？_蚂蚁淘，【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城
欢迎来到蚂蚁淘！（生命科学科研用品平行进口B2B商城） 请登录 免费注册 询价篮
我的蚂蚁淘
产品分类
网站导航
微信关注
联系QQ
0
热门关键词 jackson二抗 Polyscience 23966 Liposomes脂质体 Biotium溴化乙锭 goldbio荧光素
我的购物车0
全部分类
酶
首页 品牌展示 产品查询 新闻资讯 帮助中心 联系我们 现货平台
商品列表 技术文章 相关问答
当前位置： > 首页 > 技术文章 >
为什么大多聚合物溶解比较慢 说明为什么大多数聚合物的溶解缓慢？
来自 : 蚂蚁淘

ANALYSISOFCELLCYCLE
MiriamCapriandDanielaBarbieri
Dept.BiomedicalSciences,Sect.GeneralPathology,
ViaCampi,287,UniversityofModena,41100Modena,Italy

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

A)Bromodeoxyuridine/PropidiumIodideTheclassicalmethodfortheanalysisofcellcycledistributionistheflowcytometricmeasurementofDNAcontentwhichcansimultaneouslydeterminetheincorporationofBromodeoxyuridine(BrdU).TheprocedurerequiresthatDNAispartiallydenaturedtoexposeincorporatedBrdUtoaspecificantibody.DenaturationisnecessarybecauseantibodiesdevelopedsofarbindonlytoBrdUinsingle-strandDNA.TheremainingundenaturedDNAisthenstainedwithPropidiumIodide(PI).Greenfluorescencefromthefluorescein-conjugatedantibodyisameasureofBrdUincorporation.RedfluorescencefromthePIisameasureofDNA.Theprotocoldescribedhereuseshigh-molarityHClforthedenaturationofDNA.FurThermore,thismethodmaybeutilizedeitherforunfixedorforfixedcellsinsUSPension.

B)Cyclins/PropidiumIodideCyclinsarekeycomponentsofthecellcycleprogressionmachinery.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/PropidiumIodideOneofthemostusedprotocolforthedeterminationofapoptosisinthedifferentphasesofcellcycleistheenzymaticinsitulabelingofapoptosis-inducedDNAstrandbreaks(TUNEL).Terminaldeoxynucleotidyltransferase(TdT)havebeenusedfortheincorporationoffluorescein-labelednucleotidestoDNAstrandsbreaksinsitu.DNAcontentisrevealedbyredfluorescencefromPI.Inordertohavemoredetails,seetheChaptersrelatedtoTUNELtechnique.

D)F-Actin/PropidiumIodideTheanalysisofapoptoticcellsandestimationoftheircellcyclespecificityisalsopossibleusingarecentmethod.ThisisbasedonidentificationofapoptoticcellswhichhavemodifiedtheircytoskletonandtheirDNAcontent.Inspecific,paraformaldehyde(PFA)fixationfollowedbystainingofF-actinwithfluorescein-conjugatedphalloidinandofDNAwithPI,areused.Furthermore,thisproceduremaybeutilizedalsoforadherentcells.

2.PROTOCOLS

A)BrdU/PIPROTOCOL
    A.2.1MaterialsBrdU(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.3.1BackgroundinformationInthisprocedurefixedcellsby4%PFAinPhosphateBufferSaline(PBS)canbeutilized.InthiscasetowashcellsonceinPBSbeforetostartatstep1isnecessary.

Moreover,bothdirectandindirectimmunofluorescencecanbeused.TheBrdUincorporationismoreevidentusingtheindirectmethod.

    A.3.2AnticipatedresultsItisrecommandedtoperformeachexperimentusinganegativeandapositivecontrolsample.Thenegativesample,inordertohaveacorrectsettingofinstrument,isassessedfollowingallthestepsexceptstep8.Thepositivesample,inordertomakesurethatthemethodworks,isassessedbyusingaproliferatingcellline(suchasU937,K562,MOLT4,etc.)followingallsteps.
A.3.3TimeconsiderationsTheprotocolissimplybutitrequireaquitelongtime.Indeed,forfewsamples,moreorless4hoursarerequired.Thedurationofthemethodisobviouslydependingonthenumberofsamples.A.3.4Keyreferences1.Dolbeare,F.,Gratzner,H:,Pallavicini,M.,Gray,J.W.1983.Proc.Natl.Accad.Sci.U.S.A.80:5573.

B)Cyclins/PIprotocol

B.2.1MaterialsPFA(A2),TritonX-100(A2),PBS,mouseserum(A2),mAbsanti-cyclins(A2),goat-anti-mouse-FITC(A2),PI(A2),GM+EDTAbuffer(A1).

B.2.2.Methodology
FIXATION

InthecaseofE,AandB1cyclins,cells(2x106/mL)arefixedby70%ethanol:

a)putallthereagentsinice;b)countandcentrifugecellsat375gfor5min;c)resuspendaccuratelythepelletin1mLGM+EDTAbuffer;d)addgently3mLof96%ethanolvortexingsamplesandworkinginice;e)storethefixedsamplesat4°C.

InthecaseofDcyclins,cellsarefixedby1%methanol-freeformaldehyde:

a)putallthereagentsinice;b)countandcentrifugecellsat375gfor5min.;c)resuspendgentlythepelletin1mLof1%formaldheydeinPBSfor15mininice;d)washinice-coldPBSasinstepb;e)add1mLof70%ethanol;f)storethefixedsamplesat4°C.

STAINING

1.Washwithice-coldPBS.

2.Resuspendthepelletin1mLof0.25%TritonX-100inPBS,vortexingsamplesandworkinginice(for5mininthecaseofD,AandB1cyclins,for10-20mininthecaseofEcyclin).

3.Washinice-coldPBSasinstepb.

4.Incubatecellswith150mLofmAbanti-cyclin(diluitedattheconcentrationof2.5mg/mLinPBS+1%ofnormalgoatserum)overnightat4°C.

5.Asinstep3.

6.Incubatefor1hourwith150mLsecondarymAbgoat-anti-mouse-FITC(1.3mg/mL)diluited1:50inPBS+1%normalgoatserum.

7.Asinstep3.

8.Incubatefor4hoursatroomtemperatureandinthedarkwith1mLof2.5mg/mLPIinPBS(+12.5mLof1mg/mLRNAsi)orincubateovernightat4°Cwith0.8mg/mLPIinPBS(+12.5mLof1mg/mLRNAsi).

9.Analysewithflowcytometerequippedwitha488nmargonlaser.

B.3.COMMENTARY

B.3.1BackgroundinformationThecriticalstepsinthemethodologyarecellfixation,permeABIlizationandtheconcentrationsofanti-cyclinmAbs.Formostcyclinsoptimalfixationis70%ethanol.Thistreatmentpreservescyclins,loweringthebackground,non-specificcellfluorescenceandresultinginanimprovedsignal-to-noiseratioofthecyclinspecificfluorescence.DetectionofDcyclin,however,requiresfixationinformaldehyde.Asfarasanti-cyclinmAbconcentrationisconcerned,2.5mg/mLisoptimalformostcells.Anyway,totestthebestconcentrationforeachexperimentalmodel,isrecommended.

B.3.2AnticipatedresultsInthisprocedure,anegativecontrolsample,whichcontainsonlythesecondaryFITC-mAb,isnecessary.

B.3.3TimeconsiderationsTheprotocolrequirearound2hoursbeforetheovernightincubationand5hoursafter.

B.3.4Keyreferences

1.Darzynkiewicz,Z.,Gong,J.,Juan,G.,Ardelt,B.,Traganos,F.1996.Cytometryofcyclinproteins.Cytometry.25:1.

2.Faretta,M.,Bergamaschi,D.,RonzoniS.,D’Incalci,M.,Erba,E.1997.DiferencesincyclinB1expressionincellcycleblockedintheG2/Mphaseaftertreatmentwithanti-canceragent.Anewthreeparametricflowcytometryanalysis.ProceedingsoftheXIVNationalItalianMeetingofCytometry.

3.Gong,J.,Traganos,F.,Darzynkiewicz,Z.1993.SimultaneousanalysisofcellcyclekineticsattwodifferentDNAploidylevelsbasedonDNAcontentandcyclinbmeasurements.CancerRes.53:5096.

4.Gong,J.,Li,X.,Traganos,F.,Darzynkiewicz,Z.1994.ExpressionofG1andG2cyclinsmeasuredinindividualcellsbymultiparameterflowcytometry:anewtoolintheanalysisofthecellcycle.CellProlif.27:357.

5.Gong,J.,Traganos,F.,Darzynkiewicz,Z.1995.DiscriminationofG2andmitoticcellsbyflowcytometrybasedondifferentexpressionofcyclinsAandB1.Exp.CellRes.220:226.

6.Widrow,R.J.,Rabinovitch,P.S.,Cho,K.,Laird,C.H.1997.SeparationofcellsatdifferenttimeswithinG2andmitosisbycyclinB1flowcytometry.Cytometry27:250.

C)TUNEL/PIprotocol

C.2.1Materialsformaldehyde(A2),ethanol,reactionmixture(A1),TdTbuffer(A1),Bio-16-dUTP(A2),TdTenzyme(A2),stainingbuffer(A1),SSCbuffer(A1),BLOTTO(A2),Avidin-FITC(A2),TritonX-100(A2),PI(A2),DNAasebuffer(A1).

    C.2.2.Methodology1.Cells(5x105-1x106cell/sample)arewashedtwiceat260gfor5minusing2mLofPBS(pH7.2).
2.Fixby1mLof1%formaldehydeinPBS,onicefor15min.3.Washonceasinstep1.4.Resuspendin1mLofice-coldethanol70%(atthispointtostorethesamplesat-20°Cfor18hoursorovernightispossible).5.Asinstep3.6.Resuspendin50mL(foreachsample)ofthereactionmixture,whichispreparedduringthelastspindown.7.Incubatefor30minat37°Cwaterbath.8.Asinstep3.9.Resuspendin100mL(foreachsample)ofthereactionstainingbuffer,whichispreparedduringthelastspindown.10.Incubatefor30minatroomtemperatureinthedark.11.Asinstep3.12.CounterstainDNAwith5mg/mLofPIinPBS.13.Incubatefor15minat4°Cinthedark.14.Analysewithflowcytometerequippedwitha488nmargonlaser.

C.3.COMMENTARY

C.3.1BackgroundinformationThisprocedureiscomplexandnotalwaysgoodresultsareobtained.Thus,theuseofcommercialkitssuchasApoTagTM(Oncor,Gaithersburg,MD,USA)and"InsitucelldeathdetectionKit"(Boeringer-Mannheim,Germany),ishighlyrecommended.

    C.3.2AnticipatedresultsToperformeachexperimentusingablank,anegativeandapositivecontrolsamples,isrecommended.Theblanksampleisassessedsubstitutingstep9asfollowing:add100mLofreactionstainingbufferpreparedwithoutAvidin-FITC.Thenegativesampleisassessedsubstitutingstep6asfollowing:add50mLofreactionstainingbufferpreparedwithoutTdTenzyme.Blankandnegativeareperformedinordertohaveacorrectsettingoftheinstrument.Thepositivesample,inordertomakesurethatthemethodworks,isassessedbydigestingwith75mLofDNAsebufferfor20minatroomtemperaturebeforestep6.
C.3.3TimeconsiderationsTheprotocolrequireaquitelongtime.Inparticular1hourandhalfbeforetheovernightincubationandacoupleofhoursafter.Obviously,utilizingcommercialkitsthedurationofmethodishighlyreduced.
    C.3.4Keyreferences1.Gorczyca,W.,Gong,J.,Darzynkiewicz,Z.1993.DetectionofDNAstrandbreaksinindividualapoptoticcellsbytheinsituterminaldeoxynucleotidyltransferaseandnicktranslationassays.CancerRes.52:1945.

    2.Gorczyca,W.,Tuziak,T.,Kram,A.,Melamed,M.R.,Darzynkiewicz,Z.1994.Detectionofapoptosis-associatedDNAstrandbreaksinfine-needleaspirationbiopsiesbyinsituendlabelingoffragmentedDNA.Cytometry15:169.

    3.Li,X.,Darzynkiewicz,Z.1995.LabellingDNAstrandbreakswithBrdUTP.Detectionofapoptosisandcellproliferation.CellProlif.28:571.

    D)F-Actin/PIprotocol

    D.2.1MaterialsPFA(A2),PBS,TritonX-100(A2),sodiumborohydride,FITC-phalloidin(A2),PI(A2).

    D.2.2.Methodology

    1.Cellsarefixedin1mLof1%PFAfor30minonice.2.Washwith0.1%TritonX-100inPBS,andincubatewith0.1%sodiumborohydrideinPBS(pH8.0)for30min.3.Washat200gfor5min.4.Incubatewith20mLofFITC-phalloidin(0.01-10.0mg/mL)for1houratroomtemperature(orovernightat4°C).5.Asinstep3.6.Resuspendedin1mLofa5-50mg/mLPIinPBSandincubatefor30minat37°C.7.Analysedwithflowcytometerequippedwitha488nmargonlaser.

    D.3.COMMENTARY

    D.3.1BackgroundinformationUsingthisprotocol,theacquisitionandanalysisofthesamplesisparticularlyimportant.Apoptoticandnonapoptoticcellsaredistinguishedonthebasisofthegreenflourescenceandthesidescatter.ApoptoticcellshavehighsidescatterandlowFL-1(1).TheanalysisofDNAcontentisrelativetothedifferentregionsofapoptoticandnonapoptoticcells.

      Inthisprocedureadherentcellscanbeutilized.Inthiscaseitisnecessarytostartthemethodasfollowing:
    i)addPFA2%directlytothecultureflasksfor30minonice.Avolumeequaltothatinthecultureflasksisadded,making1%thefinalPFAconcentration;

    ii)washat200gfor5min(continuetostep1).

    D.3.3TimeconsiderationsTheprotocolisrelativelysimpleandfast,inparticular2hoursandhalfarebasicallynecessary.

    D.3.4Keyreferences1.Endresen,P.C.,Prytz,P.S.,AarbalckeJ.1995.AnewflowcytometrymethodfordiscriminationofapoptoyiccellsanddetectionoftheircellcyclespecificitythroughstainingofF-ActinandDNA.Cytometry.20:162.

    Appendix1:Stocksolutions

    SolutionPreparationStorage
    A.Washingbuffer0.5%Tween20inPBS4°C
    A.Boraxbuffer0.1MBorax(Sodiumtetraborate-10-hydrate)RT
    A.PIbuffer3.4mMTrisodiumCitrate,9.65mMNaCl,PI20mg/ml,0.03%NonidetP-40inH2O4°C
    B.GM+EDTAbufferglucose1.1g/L,NaCl8g/L,KCl0.4g/L,Na2HPO4.2H2O0.2g/L,KH2PO40.15g/L,EDTA0.2g/L4°C
    C.reactionmixture50mLofsolutionwascomposedby:37.8mLofdeionizedwater+5mLofTdTbuffer(10X),+5mLofCoCl2(25mM),+2mLofBio-16-dUTP+0.2mLTdTenzyme0°C
    C.TdTbuffer(10X)1MNacacodylate(pH7.0),1mMdithiothreitol,0.5mg/mLserumalbumin4°C
    C.stainingbuffer100mLofsolutionwascomposedby:54.2mLofdeionizedwater+SSCbuffer(20X),+20mLofBLOTTO(25%)+0.7mLAvidin-FITC(160X),+0.1mLofTritonX-1004°C
    C.Avidin-FITC160X1mgAvidin-FITCin250mLPBS.Thendiluit1/10indeionizedwatertohave160Xstock4°C
    C.SSCbuffer(20X)0.3%sodiumcitrate,3MNaCl(pH7.0)RT
    C.DNAasebuffer20ng/mLDNAasi,10mMTRIS-HCl(pH7.4),10mMNaCl,5mMMgCl2,0.1mMCaCl2,25mMKCl0°C

    Appendix2:Reagents

    Avidin-FITCSigmaAldrichA2910
    anti-BrdUantibodyBectonDickinson347580
    Bio-16-dUTP(50nmol/50mL)BoehringerMannheim1093070
    BLOTTO(drynon-fatmilk)Bio-Rad170-6404
    BoraxRiedel-Dehaen31457
    BrdUSigmaAldrichB5002
    DNAasiBoehringerMannheim776785
    FITC-phalloidinSigmaAldrichP5282
    formaldehydeBDH10113
    goat-anti-mouse-FITCantibodyBectonDickinson349031
    AntiAcyclinantibodySantaCruzBiotechnologysc-239,sc-596,sc-751
    AntiB1cyclinantibodySantaCruzBiotechnologysc-245,sc-752,sc-595,

    sc-594

    AntiD1cyclinantibodySantaCruzBiotechnologysc-6281,sc-246,sc-450,

    sc-717,sc-753,sc-618

    AntiEcyclinantibodySantaCruzBiotechnologysc-247,sc-198,sc-481
    mouseserumCaltag10410
    NonidetP-40SigmaAldrichN6507
    paraformaldehydeSigmaAldrichP6148
    PISigmaAldrichP4170
    BovineserumalbuminSigmaAldrichB7276
    TritonX-100SigmaAldrichT9284
    TdTenzyme(25U/mL)BoehringerMannheim220582
    Tween20Merk-Schuchardt822184

    Appendix3:Equipment

    FlowCabinetTC60Gelaire
    FlowCytometerFACScanBectonDickinson
    IncubatorCO2-AUTO-ZEROHeraeus
    MinifugeRFHeraeus
    PipetmanP20,P200,P1000Gilson
    VortexVibrofixVF1ElectronicJanke&Kunkel-IkaLabortechnik
    WaterBathD8Haake

免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
发布于 ： 2021-09-21 阅读（365）
▍ 相关分类
酶
蛋白酶类 其它酶 激酶
重组蛋白
细胞因子 生长因子 人体抗原 病毒抗原 其它动物抗原 其它重组蛋白 植物抗原 细菌抗原
天然蛋白
白蛋白 球蛋白 细胞裂解和提取物 其它天然蛋白 血清纯化蛋白
蛋白纯化
蛋白纯化试剂盒 透析 超滤
聚合物
多肽
▍ 相关文章
【求助】细胞培养液变浑浊 细胞技术讨论版 专业医生社区,医学... Tubulin Preparat 实验方法 巢式PCR概述和实验操作步骤实验技术ShengSci 为什么大多聚合物溶解比较慢 说明为什么大多数聚合物的溶解缓慢？ NBA2K17面补下载合集_NBA2K17面补修改器大全_游戏下载 ... 一种培养皿固体培养基分装装置 质粒和PCR产物测序 【求助】721无法调零是怎么回事？_紫外可见分光光度计（UV）仪器... 牛结合珠蛋白/触珠蛋白(Hpt/HP)酶联免疫分析(ELISA)说明书 Tulstar Products Inc.进口数据及联系方式 轮状病毒抗原检测试剂盒(乳胶法)(艾博生物医药(杭州))说明书_功效... PremiumHD Travi Csajok
▍ 相关问答
抗eb病毒早期抗原igM抗体阴性抗eb病毒核抗原igG抗体阳..._寻医问药...
PEDV和TGEV毒株间的抗原关系
艾滋病抗原抗体什么意思
一种犬瘟热、犬细小病毒病、狂犬病三联亚单位疫苗的制作方法
细胞因子
【分享】关于发布《2019新型冠状病毒抗原/抗体检测试剂注册技术...
乙肝病毒抗原和抗体的关系_
流感病毒有多恐怖?《工作细胞》来告诉你!
甲乙流感病毒抗原检测怎么做?_即问即答_家庭医生在线即问即答
简述乙型病毒性肝炎抗原抗体系统检测的临床意义？
乙肝病毒抗原抗体系统的意义乙肝检查
巨细胞病毒早期即刻蛋白+早期基因蛋白测定, _有问必答_寻医问药网
自营商城图标
自营商城
一手货源 13%专票
正品保障图标
正品保障
全球直采 在线追溯
解放采购图标
解放采购
在线下单 简单省事
及时交付图标
及时交付
限时必达 畅选无忧
版权所有 : ©2005- 苏州蚂蚁淘生物科技有限公司
公司邮箱 : info@ebiomall.com
联系电话 : 4000-520-616

© 2005- 苏州蚂蚁淘生物科技有限公司 版权所有。 苏ICP备17049038号-11 苏公网安备 32059002001627号

声明：本网站部分产品受外界条件影响（包含但不限于进口国出口国政策限制等因素），无法提供销售服务，下单后，会由客服人员联系厂家确认后另行通知。部分危险品无法清关，请勿下单

友情链接
首页
分类
品牌中心
购物车
我的