流式細(xì)胞儀操作-37頁P(yáng)PT課件

1、流式細(xì)胞技術(shù)基礎(chǔ)主講 人 繆祥 2019.9.18流式細(xì)胞技術(shù)論壇 flowcytocoBD FACS Aria 1Beckman MoFlo AstriosBD Calibur我們擁有的流式細(xì)胞儀Beckman Gallios一、光源二、流動室三、軟件分析系統(tǒng)流式細(xì)胞儀的組成 1. 激光 目前最高的配置是7激光， 355nm 、405nm、 457nm 、488nm 514nm、 561nm 、633nm (Dream)355nm Side Population （SP）必須滴405nm 免疫多色分析 PACIFIC BLUE Brilliant violet(405)457nm 染色體分選

2、488nm GFP Alex488 PE PE-CY5 PE-CY7 TEX-RED Percp5-5 .514nm YFP 561nm Mcherry PE 最佳的激發(fā)波長633nm APC APC-CY7 Alexa 647 2. 汞燈 現(xiàn)在用的比較少了，比較大的缺點(diǎn)是能量比較低光源 流式細(xì)胞儀的光信號 散射光信號 熒光信號 -前向角散射光（FSC,Forward Scatter）入射激光的同向散射光信號，反映了細(xì)胞相對大小及其表面積 - 側(cè)向角散射光 （SSC, Side Scatter）入射激光90角的散射光信號，反映了細(xì)胞粒度及細(xì)胞內(nèi)相對復(fù)雜性熒光素吸收激光能量后將吸收能量釋放，轉(zhuǎn)換

3、為振動能和熱能釋放較入射光波長更長的光量子 前向角散射光 FSCForward angle light scatterFALSFALS SensorLaser 側(cè)向角散射光SSCFALS Sensor90LS SensorLaserFSC-SSC ApplicationWhat can we read from this fsc-ssc dot plot?1.FSC 從左往右 細(xì)胞越來越大，反應(yīng)細(xì)胞直徑3.FSCSSC 圖能夠幫助大家判斷細(xì)胞的相對大小，細(xì)胞越大FSC的電壓值越小2.SSC從下往上細(xì)胞內(nèi)部顆粒越來越多，反應(yīng)被測細(xì)胞的細(xì)胞膜、細(xì)胞質(zhì)、核膜的折射率和細(xì)胞內(nèi)顆粒的性狀熒光是怎么產(chǎn)生的

4、呢？510nm-530nm陰性細(xì)胞為何有峰圖?FITCAlexa488Alexa488Synthetic, organic dyes:Alexa Fluors, Cy dyes, Horizon, Dylight, etcProteins: PE, APC, GFP, mCherrySemiconductor nanocrystals: Qdots, Crystalplex, eFluorNCOrganic polymers: Brilliant VioletTMHigh-Sensitivity FluorescenceAlexa488 fluorescent chemistry famili

5、escoumarintryptophan350Pacific BlueAlexa 488FITCAlexa 568/ rhodamineTexas RedCy7Organic fluorophores復(fù)合染料-PE-Cy5If the flourophores have overlapping spectral properties with proximal and proper orientationThe Donor molecule (PE) transfers it energy to excite the Acceptor molecule (Cy5) instead of emi

6、tting photonsThe excited Acceptor then releases the energy at its emission wavelengthPECy5PECy5Fluorescence Resonance Energy Transfer (FRET)0 hours2 hours22.5 hoursPE(FL2)PE-Cy5PE-Cy7Time Sample Left in LightTandem Conjugates functionality can be sensitive to the environment如何選擇熒光染料 1、根據(jù)現(xiàn)有的儀器所有的激光器與

7、通道進(jìn)行選擇，不能只有488nm、633nm 的激光卻選擇405nm激光激發(fā)的熒光素 ，calibur 是雙激光4色的流式細(xì)胞儀，所包含通道FITCPEPERCP5-5APC2、表達(dá)比較弱的選擇比較強(qiáng)的熒光素，表達(dá)比較強(qiáng)的選擇較弱的熒光素 CD133一般都選擇PE MHC-II 一般選擇FITC等較弱的熒光素，在很多情況下它的表達(dá)太高 PE是常用熒光素中亮度最強(qiáng)的 FITC比較弱，比較穩(wěn)定3、在可以的情況下選擇熒光補(bǔ)償最小的熒光素，比如FITC PERCP5-5、APC4、優(yōu)先選擇直標(biāo)抗體，沒法子的時候才選擇間標(biāo) 能不選擇復(fù)合染料盡量不要選擇復(fù)合染料 比如PE-CY 5 PE-CY7 CV比較

8、大 補(bǔ)償較大5、PE-CY5盡量不與APC一起染，因?yàn)樗麄兊陌l(fā)射波長幾乎重疊FluorophoreBrightness indexabundance of Agproblematic backgroundfavorite ratingBV4215the lowesthighestBV5703-4midBleeds into PE and BV421middlePacific Blue1low mid to highhighPacific Orange1very highvery lowV5001Very highlowFITC/AF4883low to midbleeds into PEhig

9、hPerCP-Cy5.53midcrossbeam into AF700middlePE 5low to midmiddlePE-TR2-3midbleeds into PE and PE-Cy5very lowPE-Cy54low to midcrossbeam into APC and bleeds into PElowPE-Cy74lowbleeds most of all tandems into PE and crossbeam into APC-Cy7middle- lowAPC/ AF6475low to midhighAF7002mid to highmiddleAPC-Cy7

10、2midbleeds into APClow熒光染料亮度與受歡迎程度 流動室(分選) 軟件分析系統(tǒng)分析軟件1. 機(jī)器自帶分析軟件：SUMMIT2.第三方分析軟件：Kaluza， Flowjo， Facs express, CFCS3.特殊分析軟件 ：周期分析軟件Modifit , WincyleflowcytocoWhat does compensation mean?Compensation is the process by which the fluorescence “spillover” originating from a fluorochrome other than the o

11、ne specified for a particular PMT detector is subtracted as a percentage of the signal from other PMTs. overlap between FITC and PE produces photons detected by both FITC (FL1) and PE (FL2) detectors.The amount of photons from FITC fluorescence leaking into the PE detector must therefore be compensa

12、ted out. Analog Compensationex. FITCArgon LaserFL3FL1FL2%450500550600FITCCyCPETrue FL2= FL2 x% of FL1FL2PEFL1FITCTotal signal detected in FL1Unwanted signal detected in FL2roughly 15%FL2 15% FL1Unwanted signal detected in FL2roughly 15%Total signal detected in FL1Uncompensated FITCFL2-15%FL1Uncompen

13、satedCompensatedFL2-30%FL1Over CompensatedTime (msec)Signal Intensity (channel)IntensityNo. of Cells/Channel如何看流式圖散點(diǎn)圖這張圖你能看出什么？如何看流式圖直方圖雙峰單峰細(xì)胞周期Model: %G0-G1: 77.75 at 69.82 %S: 11.87 %G2-M: 10.37 at 139.65 %CV: 6.52RCS: 1.246Linear or Log AmplificationLinear amplifierLog amplifierCD8 Expression of

14、LymphocytesWhen do we use linear amplifier?Cell cycle associated assayEx: PI ; Hochest ; 7AAD Brdu-PI ; Brdu-7AADWhen do we use log amplifier?Mean 平均值 即所有細(xì)胞的熒光強(qiáng)度相加，再除以細(xì)胞數(shù)GeoMean 幾何平均數(shù) 即所有細(xì)胞的熒光強(qiáng)度相乘，再進(jìn)行N次開方，N= 細(xì)胞數(shù)Median 中位數(shù) 是對所有細(xì)胞的熒光強(qiáng)度進(jìn)行排序，位于50處的熒光強(qiáng)度值平均熒光強(qiáng)度如何準(zhǔn)備對照以三色CD4-FITC、CD8-PE、CD19-PeCy7為例，對照組如下：

15、blank：調(diào)電壓； FITC單標(biāo)：調(diào)補(bǔ)償； PE單標(biāo)：調(diào)補(bǔ)償； PeCy7單標(biāo)：調(diào)補(bǔ)償； FITC FMO對照： 加FITC-IgG、 CD8-Pe和CD19-PeCy7； PE FMO對照： 加FITC-CD4、PE-IgG和CD19-PeCy7； PeCy7 FMO對照：加FITC-CD4、PE-CD8和IgG-PeCy7；FMO ControlIso controlBlank Fluorescence Minus One (FMO) Control空染Isotype 相同種屬來源、相同亞型、相同劑量和相同的免疫球蛋白 ：CD4 fitc IgG1-fitc IgG12.當(dāng)不能確定陰性或者陽性的位置的時候需要找陰性、弱陽 性對照例如：做凋亡的時候，空白對照與