BD流式全面介紹

1、1會計學(xué)BD流式全面介紹流式全面介紹Injector TipSheath fluid1. 散射光信號散射光信號前向角散射光（前向角散射光（FSC,Forward ScatterFSC,Forward Scatter） 入射激光的同向散射光信號入射激光的同向散射光信號 細(xì)胞相細(xì)胞相 對大小及其表面積。對大小及其表面積。 側(cè)向角散射（側(cè)向角散射（SSC, Side ScatterSSC, Side Scatter） 入射激光入射激光9090 角的角的散射光信號散射光信號 細(xì)胞粒度及細(xì)胞內(nèi)相對復(fù)雜性。細(xì)胞粒度及細(xì)胞內(nèi)相對復(fù)雜性。FALS SensorLaserFALS Sensor90LS Sens

2、orLaserlysed whole bloodDead cells are known to be smaller and to exhibit more internal complexity than live cells. Which of the populations on this plot would you expect to be dead?FreqFluorescenceFALS SensorFluorescence detector(PMT3, PMT4 etc.)Which of the three populations has the most Ab A bind

3、ing sites?Ab AAb BSampleLaminarFlowLow Sample Flow Rate12 mL/minSheathSheathSampleLaminarFlowSheathSheathOptical CuvetteHigh Sample Flow Rate60 mL/minWhich of the following would cause disturbance in the laminar flow of the optical cuvette? A. bubblesB. cellular concentrationC. sample flow rate 460

4、500 540460 500 540460 500 540LongpassShortpassBandpassLaserLaserFluorochromesFluorochromesDetector Detector ParameterParameterFilterFilterTimeVoltageTimeVoltageTimeVoltageTimeVoltsPulse AreaPulse HeightPulse Width0 FL2FL1SSCFSCFL4FL3400 nm500 nm600 nm700 nmR e l a t i v e I n t e n s i t yPIExcitati

5、onEmisson300 nm 400 nm 500 nm 600 nm 700 nm400 nm500 nm600 nm700 nmR e l a t i v e I n t e n s i t yWavelength300 nm 400 nm 500 nm 600 nm 700 nm300 nm 400 nm 500 nm 600 nm 700 nmProtein300 nm 400 nm 500 nm 600 nm 700 nm補(bǔ)償調(diào)節(jié)前后對比補(bǔ)償調(diào)節(jié)前后對比FL1FL2 Fluorescent probe attached to antibody Specific signal: we

6、ak Nonspecific binding: low Fluorescent probe attached to a 2nd antibody Specific signal: strong, 5-6 2nd Ab/each 1st Ab; Nonspecific binding: highbiotinylated primary Abbiotinavidinbiotinylated dye儀器設(shè)置調(diào)節(jié)儀器設(shè)置調(diào)節(jié)1. 用未染色細(xì)胞調(diào)整儀器用未染色細(xì)胞調(diào)整儀器PMT電壓電壓2. 用單染色的細(xì)胞調(diào)節(jié)儀器補(bǔ)償用單染色的細(xì)胞調(diào)節(jié)儀器補(bǔ)償在上述信號基礎(chǔ)上的細(xì)胞分選在上述信號基礎(chǔ)上的細(xì)胞分選流式細(xì)胞術(shù)

7、的細(xì)胞學(xué)應(yīng)用流式細(xì)胞儀的遺傳學(xué)應(yīng)用細(xì)胞細(xì)胞DNA, RNA 含量的測定含量的測定染色體倍體分析染色體倍體分析染色體分離染色體分離基因表達(dá)產(chǎn)物的生物活性研究基因表達(dá)產(chǎn)物的生物活性研究基因轉(zhuǎn)染表達(dá)的生物效應(yīng)基因轉(zhuǎn)染表達(dá)的生物效應(yīng)基因表達(dá)調(diào)控研究基因表達(dá)調(diào)控研究細(xì)胞內(nèi)基因定位細(xì)胞內(nèi)基因定位酵母轉(zhuǎn)基因株的篩選酵母轉(zhuǎn)基因株的篩選報告基因的定性定量檢測報告基因的定性定量檢測植物遺傳學(xué)研究植物遺傳學(xué)研究 Cell Lineage/Subset PhenotypeCytokine/ChemokineProduction RepertoireCytokine/ChemokineReceptor Repertoi

8、reMigration/Homing PhenotypreCell cycle statusDNA 探針探針DNA探針最主要的特點(diǎn)是，它們與DNA含量是成化學(xué)正比化學(xué)正比關(guān)系的這樣，帶有的探針熒光分子的數(shù)目就和DNA分子的含量相當(dāng)，從而檢測DNA含量 Nucleic acid Probes菲啶基Propidium IodideEthidium Bromide苯甲亞胺Hoechst 33342抗生素Mithramycin, Chromamycin A3Acridine Orange - AOPyronyn Y0 200 400 600 8001000DNA contentCountA typic

9、al DNA HistogramG0-G1SG2-MFluorescence Intensity# of Events 增殖相關(guān)基因增殖相關(guān)基因PCNA(Proliferating cell nuclear antigen) 一種蛋白質(zhì)，在DNA復(fù)制和核苷酸的切除修復(fù)中起到作用Ki-67 - proliferation related antigenKi-S1 - proliferation related antigenPhospho-histoneCyclin D1, E, A, B1 Flow cytometry of apoptotic cell deathFlow Cytometry

10、 of Apoptotic CellsPI - Fluorescence# EventsApoptotic cellsNormal G0/G1 cellsPositive CellAbsolute Count BeadsNegative CellCD4 PECD4 PE HAMControlHIVCD8Tax-A2/IgGag-A2/IgTraditional Cytokine Flow CytometryIL-2 PhycoerythrinCD4 FITCAllows the correlation of: Phenotype Cytokine expressionCell CyclePro

11、liferationPPYYYY非磷酸化特異性抗體檢測全部的相關(guān)蛋白 unphosphorylated + phosphorylated亞型 抗原：重組的蛋白質(zhì)片斷磷酸化特異性抗體 僅僅檢測磷酸化亞型 抗原：合成的 4-10mer磷酸化肽段Y磷酸化特異性和非磷酸化特異性抗體的差異BD PhosFLOW和和Western blot 的比較的比較A theoretical experiment comparing Western blot and flow cytometry with three samples and a protein of interest at 1, 10, or 50

12、copies per cell. Sample 2 and 3 look the same via Western blot, but when stained with fluorescently labeled antibodies, the differences between the samples become more relevant. (Source: P.O. Krutzik et al. / Clinical Immunology 110 (2004) 206221)BD PhosFlowGenomics & Proteomics - Single Cells H

13、ave Big Proteomics Story to Tell. BD PhosFlow not only tells you activation of a single cell for one particular phospho protein and pathway, but allows you to study multiple phosphorylation events and pathways simultaniously.100101102103104100101102103104PBMC050100150200250FSC-H050100150200250SSC-HC

14、D20 PerCP-Cy5.5CD3 PE Zap70 (Y319)/Syk (Y352) Alexa 647 100101102103104020406080100100101102103104020406080100100101102103104020406080100100101102103104100101102103104CD3 PE 100101102103104020406080100100101102103104020406080100100101102103104020406080100CD3-/CD20+ CD3+/CD20- CD3-/CD20-CD20 PerCP-Cy

15、5.5Whole Blood CD3 CrossLink 050100150200250FSC-H050100150200250SSC-H21.8Multi-Color PhosFlow in CD3/CD28 or CD3 crosslinked human PBMCs or Whole BloodCD3/CD28 CrosslinkUntreated cells (unshaded) vs treated cells (shaded)Shades of a colorAntibody coupled beads, emitting at distinct FL3 intensitiesVa

16、rious analytesAntibody coupled PE label, emitting at FL2 intensity proportional to analyte conc.0 pg/mL80 pg/mL1250 pg/mL5000 pg/mLFL3 Beads FL2 (PE) 從單一小體積樣本中得到多項檢測結(jié)果 對所有的檢測項目，只需要制備一個標(biāo)準(zhǔn)混合物來繪制標(biāo)準(zhǔn)曲線 避免人為造成的酶聯(lián)放大的假陽性信號產(chǎn)生 用更少的時間和精力，卻得到大量的結(jié)果 如果是用488nm和635nm兩根激光器來實(shí)驗，實(shí)驗條件更容易建立 使用配有HTS選件的BD流式細(xì)胞儀可以做到自動平板上樣和高通

17、量檢測ABCDEFGHI1 2 3 4 5 6 7 8 9FL4-H or Red-AFL3-H or NIR-AOld Format BD CBA BeadsBD CBA Flex Set BeadsABCDEFGHI1 2 3 4 5 6 7 8 9FL4-H or Red-AFL3-H or NIR-A1234567891. Itk (Y511)2. ERK (T202/Y204)3. JNK (T183/Y185)4. P38 (T180/Y182)5. PLCg g (Y783)6. ZAP70 (Y319)7. LAT (Y171)8. c-Jun (S63)9. RSK (S38

18、0)110100FOLD INCREASE IN UNITS/ML05101520TIME (MINUTES)RSK (S380)Jun (S63)LAT (Y171)Itk (Y511)ZAP-70 (Y319)PLCg g (Y783)P38 (T180/Y182)JNK (T183/Y185)ERK (T202/Y204)P-Specific AntibodiesLckZAP-70PItkPPMAPKKKPIKKPNF-kBRasGrb2SOSRacPPPLCPPDAGPKCPVAVSLP-76PLATPPPPPP PPPPPPRasMEKERKJNKp38MKK4,7MKK3,6MEK

19、K1-4ElkJunATF2MEKK4LATPPRSKSpecificitySpecificity UnstimulatedUnstimulatedAnti-CD3Anti-CD3Change Change % of Control% of ControlZAP-70108763655100Itk3711477100LAT42212170100PLCg609881272100ERK10430502946100JNK131272141100p3852729252398100c-Jun209351142100RSK55440385100Cells were activated with anti-

20、CD3/CD28 for 2 minutes. SDS was added to a final concentration of 1% and the material was placed in a boiling water bath for 5 minutes.LckZAP-70PItkPPMAPKKKPIKKPNF-kBRasGrb2SOSRacPPPLCPPDAGPKCPVAVSLP-76PLATPPPP PPPPPPRasMEKERKJNKp38MKK4,7MKK3,6MEKK1-4ElkJunATF2MEKK4LATPRSKSpecificitySpecificity Unst

21、imulatedUnstimulatedAnti-CD3Anti-CD3ChangeChange% of Control% of ControlZAP-70941064970148Itk391006179LAT2518215792PLCg61774212546ERK4541737213JNK1302229265p384172109169271c-Jun204399195137RSK404992Jurkat cells were pre-incubated with 200 mM PD-98059 (MEK inhibitor) for 20 minutes before being activ

22、ated activated with anti-CD3/CD28 for 2 minutes. SDS was added to a final concentration of 1% and the material was placed in a boiling water bath for 5 minutes.LckZAP-70ItkMAPKKKIKKNF-kBRasGrb2SOSRacPLCDAGPKCVAVSLP-76LATRasMEKERKJNKp38MKK4,7MKK3,6MEKK1-4ElkATF2MEKK4LATRSKJunSpecificitySpecificity UnstimulatedUnstimulatedAn