流式细胞术整体解决方案

Attune流式细胞仪|Bigfoot全光谱分选仪|18,000+|80+种荧光染料|PrimeFlow流式RNA流式细胞术能够从单细胞水平同时分析细胞样本中的基因完成流式实验通常需要将多种抗体搭配成多色流式实验方案。这本流式细胞术整体解决方案手册不仅介绍了Invitrogen™eBioscience™Invitrogen™流式细胞Invitrogen™Attune™入地了解异质性细胞群的详细信息。无论进行细胞亚群鉴••炎症•••••流式细胞术整体解决方案实验流程确定免疫分型抗体样品制备选择缓冲液细胞功能检测实验对照上机检测活细胞死细胞流式细胞术实验流程。2免疫细胞通常需要刺激活化后才能快速增殖或分化为成熟•Invitrogen™eBioscience™式细胞术进行检测。选择合适的激活剂/刺激剂需要根据(3)•胞培养•ACD28receptorBIonomycinCConADLPSEM-CSFCD3receptorPMACD28/CD3TCR介导的激活和共刺激IonomycinandPMA激活并刺激白细胞ConA丝裂原介导的白细胞激活LPS激活白细胞M-CSF巨噬细胞和单核细胞的分化和存活图2.(A)CD3CD28(B)eBioscienceCellstimulationcocktailPMA-瘤素（-4（4）。(C)Con(D)脂LPS-6（6（子(E)TCD4T细胞群内鉴定人类细胞TPMA和离子霉素或CD3/CD28功能级抗体刺CD4–FITC图3.CD4+TeBioscience500×Invitrogen™eBioscience™Invitrogen™抗人CD4APC和Invitrogen™eBioscience™PE-eFluor™标记的抗人320+Invitrogen™流式抗体连接了80+Invitrogen™•••荧光染料亮度•CD标志物•••选择靶标抗原•18,000+种Invitrogen™站上流式抗体的搜索结果中包含内部抗体验证*测试数据和StepInvitrogeneBioscienceStep图4.Step2eFluor450APCAPCAPC-eFluor78010075NKTcells445025000000300400500600700800900300400500600700800900CD3APC-eFluor450CD3APC-eFluor780波长（nm）波长（nm）图5.Anti-humanCD3Invitrogen™eBioscience™APC-eFluor™780InvitrogeneBioscienceeFluor450Anti-humanCD56-APC问™4Invitrogen染料家族类型优势Invitrogen荧光染料天然染料••FITCInvitrogenPaciꢀc系列染料部分染料的亮度很低PaciꢀcBluePaciꢀcOrangeAlexaFluor405AlexaFluor488AlexaFluor532AlexaFluor561eFluor450AlexaFluor647AlexaFluor660AlexaFluor700有机染料：稳定小分子••以其激发光波长命名InvitrogenAlexaFluor覆盖整个光谱的稳定染料系列染料••可用于流式细胞术和成像实验InvitrogeneBioscienceeFluor660eFluor有机染料eFluor506•••用于流式细胞术APC(allophycocyanin)PerCP(peridininchlorophyllprotein)PE(phycoerythrin)天然染料部分染料的亮度很高APC-Cyanine5PE–AlexaFluorPE–AlexaFluor700APC–AlexaFluor750PE–eFluorAPC-Cyanine7大分子蛋白染料PE-Cyanine5(TRI-COLORdye)PE-Cyanine5.5•供体染料与受体染料的发射光谱不偶联染料PE-Cyanine7PerCP–eFluorAPC–eFluor780PE–TexasReddyePerCP-Cyanine5.5•••405nm紫激光激发SuperBright436SuperBright600SuperBright645SuperBright702SuperBright780荧光溢漏更少InvitrogeneBioscienceSB染料时需加入InvitrogeneBioscienceSuperBrightCompleteStainingBuffer(货号SB-4401-42)聚合物染料SuperBright系列染料BrilliantUltraViolet395BrilliantUltraViolet496BrilliantUltraViolet563BrilliantUltraVioletBrilliantUltraViolet661BrilliantUltraVioletBrilliantUltraViolet805BrilliantVioletBrilliantViolet480BrilliantViolet605BrilliantViolet650BrilliantVioletBrilliantViolet786••由紫激光或紫外激光激发InvitrogeneBioscienceBrilliantViolet和BrilliantUltraViolet系列染料聚合物染料流式新品BUV染料时需加使用两种或两种以上入Invitrogen™BrilliantStainBuffer(00-4409-75)••发射光谱窄InvitrogenQdot系列Qdot605Qdot655Qdot705Qdot800Fluorescence染料resonanceenergytransfer,FRET也NovaFluorBlueNovaFluorYellow660NovaFluorYellow690NovaFluorYellow700NovaFluorYellow730NovaFluorYellow755NovaFluorRed660NovaFluorRed685NovaFluorRed700NovaFluorRedNovaFluorRedNovaFluorRed755NovaFluorBlue530•••••独特的光谱特征NovaFluorBlue555以其激发光和发射光波长命名NovaFluorBlue585NovaFluorBlue610-30SNovaFluorBlueNovaFluorBlue660-40SNovaFluorBlue660-120SNovaFluorBlue690DNA结构创新染料InvitrogeneBioscience流式新品NovaFluor系列染料在使用NovaFluor染料标记细胞时，使用InvitrogenCellBlox单核B001T03F01NovaFluorYellowNovaFluorYellow590NovaFluorYellow5Invitrogen™PanelBuilderPanelBuilder•PanelBuilder•谱重叠•Excel™StepStep2:Step3:Step4:Step5:选择流式仪器配置输入抗原指标选择荧光染料生成实验方案图6.InvitrogenPanelBuilder简单赛默飞科学家流式配色••••400-820-8982/800-820-8982LifeScience-CNTS@thermofiBuilder，6NovaFluorNovaFluor•光谱更窄—NovaFluor•亮度可选—NovaFluorNovaFluorBlue660/40S和NovaFluorBlueCellBlox封闭缓冲液的作用是避免NovaFluor染料与部分细CellBlox封闭NovaFluorCyanine染细胞这些细胞之间的非特异性结合，从而降低背景荧光信•荧光稳定—NovaFluor•特异性强—CellBloxNovaFluor染料与表2.NovaFluor*CD4-SK3荧光标记nm）nm）nm）激nm）荧光扩散NovaFluorBlueFITC496494509494494509509509509494482552496552552552496552552552552496650637637639639636636520530555585665665690695568590663667690700755785660659685700755(498-518)B2(516-533)B2(516-533)B3(533-550)B4(571-590)B6(605-625)B6(605-625)(652-669)(652-669)B9(688-707)B9(688-707)(567-587)(567-587)YG2(588-608)YG3(605-625)YG4(652-669)YG5(669-687)YG6(706-735)(706-735)YG8(735-765)YG9(765-795)(652-669)R2(669-687)R3(688-707)R3(688-707)R4R5R64884881,85669155NovaFluorBlue530NovaFluorBlue555NovaFluorBlue585NovaFluorBlue610/30SNovaFluorBlue610/70SNovaFluorBlue660/40SNovaFluorBlue660/120SNovaFluorBlue690PerCP-Cy5.54884884882,3433,3845,2264,992948830488488488488488NovaFluorYellowPE56152488;56156125496NovaFluorYellow590NovaFluorYellowNovaFluorYellow660PE-Cy55614,2576,8243,2999,8443,7893,3014,256561488;561561540190352363108234236NovaFluorYellow690NovaFluorYellow700NovaFluorYellow730NovaFluorYellow755PE-Cy7561561561488;561640APCNovaFluorRed660NovaFluorRed685NovaFluorRed700NovaFluorRedNovaFluorRedNovaFluorRed755640640640640637637*光谱流式数据均由激光Cytek™Aurora™SpectroFlo™7/Invitrogen™•••3.CD*4.流式实验缓冲液选择指南染色缓冲液位置eBioscience流式染色缓冲液细胞表面细胞胞浆00-4222）eBioscience剂套装(88-8824)eBioscienceFoxp3/转录00-5523）DNA*问8利用流式可以检测哪些方面的应用？细胞增殖细胞周期细胞凋亡细胞活性细胞代谢细胞群倍增活细胞Annexinconjugates,可固定线粒体感受器CellEventCaspase-3/7GreenDetectionReagentMitotrackerDyesreagentsVybrantDyeCyclestainsLIVE/DEADstains,eFluorfixableviabilitydyes线粒体探针MitoProbeTMRM,JC-1,MitoProbeDiOC(3),MitoProbeDNA复制固定细胞非固定2DilC(5),MitoProbe1Click-iTPlusEdU,FxCyclestains,CellEventSenescence,Propidiumlodide,stains,7-AADstain,DRAQ7dye,kitsDNAreplicationClick-iTEdU,BrdUreagents氧化应激7-Aminoactinomycin(7-AAD)dyesPropidiumlodideROSAssay,CellROXstains,HypoxiaGreenreagent钙指示剂eBioscienceCalciumSensorDyeeFluor514Indo-1AM表5.死细胞。Invitrogen™细胞活性检测试剂均采用一步法或二激光器非固定染料可固定染料UVDAPILIVE/DEADFixableBlue(450)LIVE/DEADFixableVioletLIVE/DEADFixableLime(506)LIVE/DEADFixableAqua(526)LIVE/DEADFixableYellow(575)LIVE/DEADFixableGreen(520)LIVE/DEADFixableOlive(557)405nmSYTOXBlue(480)SYTOXGreen(523)EV488nm561nmPropidiumIodideSYTOXAADvanced(647)LIVE/DEADFixableRedSYTOXOrange(570)LIVE/DEADFixableOrange(602)SYTOXAADvanced(647)•Invitrogen™™dyeLIVE/DEADFixableFarRed(665)LIVE/DEADFixableScarlet(723)LIVE/DEADFixableNearIR(775)LIVE/DEADFixableNearIR(780)LIVE/DEADFixableNearIR(876)633/5nmSYTOXRed(660/20*)•Invitrogen™Vybrant™CM-DiI(ꢀxable)DiO和DiDdyesVybrant™盒808nm最大发射波长(nm)•Invitrogen™Di-8-ANNEPSdyes9LIVE/DEADABC在使用细胞活性染料去检测固定后的细胞时，请选择LIVE/DEADInvitrogen™LIVE/DEAD™可固定细胞活性染料在细胞固定和/或破膜后仍能准确地评估细胞活性（图Invitrogen™Attune™CytPix™活细胞死细胞图8.使用LIVE/DEAD(A)Jurkat细胞用LIVE/DEADLime506染色(B)(C)BAttuneCytPix•••Invitrogen™LIVE/DEAD™BacLight™9图9.使用LIVE/DEADBacLightAttuneCellTrace试剂进行传代追踪Invitrogen™CellTrace™试剂通过分析在连续传代后细胞亚群中的染料稀释浓度来追踪细胞分裂代数（图10细胞发生增殖时，子细胞的荧光亮度是上一代母细胞的一CellTrace增殖分析实验通常检测平均DNA含量或者细胞代谢指标，DNA表6.检测原理应用Click-iTPlusEdU流式检测试剂盒DNA中是是细胞增殖BrdUDNA中是是细胞增殖CellTraceKi-67MCM2体PCNA体是是是是是是否否544127140VioletViolet-AndrewISACSRL10010¹10²10³104105CellTraceViolet荧光染料使用CellTrace试剂检测细胞增殖。使用Invitrogen™CellTrace™VioletInvitrogen™CD3RNAInvitrogen™PrimeFlow™RNA流式检测允许流式细胞术同时检测数百万个单细胞内的RNAPrimeFlow技术原理将荧光原位杂交（FISH链DNAbDNA）信号放大专利技术（图用Invitrogen™AlexaFluor™AlexaFluor™AlexaFluor™和AlexaFluor™时检测单个细胞中多达4种RNA•RNA•RNA••mRNA•检测非编码RNARNAmiRNARNAlncRNA）•RNA载量•检测端粒DNAF4/80eFluor450F4/80eFluor450F4/80eFluor450F4/80eFluor450F4/80eFluor450使用PrimeFlowRNAαmRNAArg1mRNACxcl13mRNA和mRNA使用PrimeFlowRNA盒对C57Bl/6Invitrogen™eBioscience™F4/80eFluor450PE-Cyanine7PrimeFlowRNAInvitrogen™αAlexaFluorArg1AlexaFluor488AlexaFluor6AlexaFluor568型4RNAF4/80-F4/80+SPMαmRNALPMmRNAArg1mRNA。荧光标记探针在细胞中加入标记探针LE）()()2RNACD8PE-Cyanine7PrimeFlowRNA流式检测实验流程。20-40问证实验结果准确性。特别是在细胞样品非常稀少或者待测重组人IgG1CD20（）IgG同型对照小鼠）小鼠IgG2a）小鼠IgG2b）小鼠IgM）kappa同型对照新一代补偿微球使用Invitrogen™UltraCompeBeads™Plus流式实验结果有着更加精准的补偿调节。当荧光标记的抗体添加到微球中，可以观察到阳性细胞群和阴性细胞群清IgG2akappa，同型对照大鼠IgG2b）大鼠IgG2c）大鼠IgM）UltraCompeBeadsPlus•）•355nm405nm）大鼠IgG2aCD21/CD35）488nm532nm绿nm叙利亚仓鼠）红633-640nm亚美尼亚仓鼠）•Invitrogen™eBioscience™SuperBrightBrilliantViolet或BrilliantViolet786UltraCompeBeadsPlus补偿微μgInvitrogen补偿微球UltraCompeBeadsUltraCompOneCompAbC总抗体补偿微球ArC胺反应补偿微球GFPBrightCompPlus补偿微球eBeads补偿微球eBeads补偿微球试剂盒*试剂盒eBeads微球GFP3种不同应用免疫表型分析细胞活性检测强度的类GFP表达和LIVE/DEAD可固定细胞种属反应性GFP亚型活性染料*管阳性微球；管阴性微球紫外光到633nm据SB染料和BV兼容多数标准激兼容多数标准激光激光器兼容性633nm488nm633nm或紫激光规格货号25Tests100Tests25tests01-3333-4101-3333-4201-2222-4101-2222-42A10346绝对计数微球4002000细胞绝对计数是一种定量检测样品中的细胞CountBrightPlus微球料。CountBrightPlus微Invitrogen™APC-eFluor™780808nm840/20nmInvitrogen™+•）•–10³010³104105106表8.Invitrogen绝对计数微球LIVE/DEADBacLight细菌活性及计数试剂盒**CountBrightPlus微球*AccuCheck微球•样品细胞浓度••细菌活性•测量参数样品类型微球大小样品细胞浓度任何类型4µm微球A6.40µm微球B6.36µm6µm微球A488nm575-585nm微球B635nm660-680nmUV-800nm385-860nm488nm498nm光谱范围C36995PCB100L34856*InvitrogenCountBright**仪器质控校准微球和细胞大小校准微球仪器的质控校准是获得准确实验结果的前提，这里的质控校准微球正是适用于确定ERF微球有3表9.Invitrogen质控校准微球AccuCheckERFVirocheckERFAlignꢁow3400-470nm外AccuCheckERF,nmViroCheckERF,390-910nm400-680nmnm激645-680nmAccuCheckERF:3.2µmViroCheckERF:nm,200nm,500nm6µm6µmµm-3.4µmA34305µmµmAccuCheckERF:A559506.0ViroCheckERF:CytKickAttune流式细胞仪搭配Invitrogen™CytKick™或CytKick™Max自动进样器，将精度与性能完美结合。上样速度更快的同时，能达到更高灵敏度，且无需担心因仪器堵塞而造4种激光和个检测参数；AttuneCytPix流式细胞仪还搭载了高速明场相机和自动图像分析••流式和成像双重结果：仅能获得多色流式数据，还能提供细胞形态学等图像信•采用抗堵设计的液流系统和稳定可靠••6•简化样品制备流程：•FDACFRpart您的实验流程Attune属性参数AttuneCytPix成像型流式细胞仪紫激光405488637Attune流式细胞仪紫激光405488532，激nm）黄激光637滤光片激光器405nm<50nsN/AN/AN/A液流系统脉冲激光功率脉冲宽度流动室石英杯凝胶耦合200×200µm样品分析体积自定义进样速率样品进样模式荧光灵敏度荧光分辨率数据采集率最大光电转化速度20µL4mL12.5-1,000µL/分钟FITC≤80PE≤30APC≤70MESF<3%35,000个细胞/秒；3465,000个细胞/前向角和侧向角散射光灵敏度侧向角散射光可检测0.2μmBangsLaboratories亚微米微球校准试剂盒。μmAttune流式细胞仪中标准0.5mmInvitrogenAttune488/10滤0.025µm过滤的Attune:44886214449791或像素分辨率0.3µm/像素20xN/AN/AN/AN/AN/A物镜放大倍率）理论分辨率0.450.6µm800nm微粒直观观察到3,000-6,000张图像/秒图像采集速率N/A仪器视野训练模型96×96像素至248×248像素29×29µm×µm白细胞和微球N/AN/A参数/特征N/A图像分析软件功能图像分析速度5-20µmN/AN/A1000张/问Invitrogen™Bigfoot™全光谱流式细胞分选仪搭配Sasquatch软SQSBigfoot全光谱细胞分选9个激光器和60Bigfoot•/epseps•6路分选到试管，4路分选到96孔板和384直下式分选到1536孔板，并提供多种上样选项和温控装•eps高•II级生物安全柜和气溶胶管理系AMS•Bigfoot全光谱超高速流式细胞分选仪参数参数激光器349nm,405nm,445nm,488nm,532nm,nm,594nm,640nm,and785nm349nm405nm445nm200488nm532nmmWnmmW594nmmW640nmmW785nmmW间光束校准检测器参数散射光分辨率55标准FSC和SSC488nmFSC405nmFSC和SSC488nm）<0.2µmAPC均MESF光谱矩阵相似矩阵SasquatchSelectedFluorescentListBabraham研究所在2020年BigfootBigfootBigfoot天可以为我们实验室节省20BigfootBigfootRachaelWalker,BabrahamBabraham•样本••Invitrogen™PanelBuilder流式配色工具结合光谱流式特征和流式配色原5您设计30色50InvitrogenPanelBuilderPanelBuilder3400-820-8982/800-820-8982LifeScience-CNTS@thermofi••推荐将AlexaFluorPaciꢀcOrangeeFluor450和SuperBright436•60+种Invitrogen™NovaF