bd流式全面介紹

1、流式細(xì)胞術(shù)簡介,For FCM Training,流式細(xì)胞術(shù),流式細(xì)胞術(shù)（Flow Cytometry, 簡稱FCM）是一種可以快速、準(zhǔn)確、客觀，并且同時檢測單個微粒（通常是細(xì)胞）的多項特性的技術(shù)，同時可以對特定群體加以分選研究對象為生物顆粒，如各種細(xì)胞、染色體、微生物、及人工合成微球等研究的微粒特性包括多種物理及生物學(xué)特征，并加以定量,流式細(xì)胞儀系統(tǒng),簡介通過流式細(xì)胞儀我們可以得到以下信息 -相對細(xì)胞大小

2、- 相對細(xì)胞顆粒密度和內(nèi)部復(fù)雜度 - 染色過細(xì)胞的相對熒光強度,,流式細(xì)胞儀的光信號,散射光信號熒光信號,1. 散射光信號,前向角散射光（FSC,Forward Scatter） 入射激光的同向散射光信號 細(xì)胞相 對大小及其表面積。 側(cè)向角散射（SSC, Side Scatter） 入射激光90?角的散射光信號 細(xì)胞粒度及細(xì)胞內(nèi)相對復(fù)雜性。,,前向角散射光 ——FSC,Forward Angle Li

3、ght Scatter,側(cè)向角散射光——SSC,,散射光,散射光能被用來區(qū)分不同細(xì)胞群體的基本形態(tài)上的差異 -通常使用“散點圖”來看散射光信號 -散點圖上的一個點就代表一個細(xì)胞顆粒的數(shù)據(jù),散點圖——Dot Plot,,lysed whole blood,Review Question,Dead cells are known to be smaller and to exhibit more internal com

4、plexity than live cells. Which of the populations on this plot would you expect to be dead?,,,,A,B,2. 熒光信號,熒光素吸收激光能量熒光素將吸收能量釋放，轉(zhuǎn)換為振動能和熱能釋放較入射光波長更長的光量子熒光素與特異抗體結(jié)合熒光抗體與細(xì)胞抗原結(jié)合越多，產(chǎn)生的熒光信號越強,熒光檢測器,,Two-Color Cell Analy

5、sis,Which of the three populations has the most Ab A binding sites?,,Ab A,Ab B,現(xiàn)代流式細(xì)胞儀包括,液流系統(tǒng) 聚焦細(xì)胞以供檢測光學(xué)系統(tǒng) 激發(fā)和收集光信號 電子系統(tǒng) 將光信號轉(zhuǎn)化為電信號，并使其數(shù)字化以供計算機分析,液流系統(tǒng),液流系統(tǒng)將樣本懸液聚焦在光源的中心處,Sample Flow in Optical Cuvette

6、,Sample,,,,,,LaminarFlow,Low Sample Flow Rate12 mL/min,Sheath,Sheath,,,,,,,,,Sample,,,,,,,,,,,,,,,,,,,,,,,LaminarFlow,,,Sheath,Sheath,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Optical Cuvette,,,High Sample Flow Rate60 mL/min,,,,,

7、,,Review Question,Which of the following would cause disturbance in the laminar flow of the optical cuvette? bubblescellular concentrationsample flow rate,Optics,Excitation optics consist of:LasersLenses and mirrors

8、 that route the laser light to the fluidic streamCollection optics consist of:Filters that direct the signals to the appropriate optical detectors,,,,Optical Filters,460 500 540,460 500 540,460 500 5

9、40,,,,,,,,,,,,,,,,,,,SP 500,,,,LP 500,,,,BP500/50,LongpassShortpassBandpass,,,,FACSCalibur光路圖,Optics,488 nm,635 nm,,,,Electronics,Converts analog signals to proportional digital signals Computes Height for each pulse

10、Calculates width and area Interfaces with the computer for data transfer,Creation of a Voltage Pulse - Analog Signal,Quantification of a Voltage Pulse,Height is a measurement for all parameters.Width = Area/Height,Effe

11、ct of the Instrument Controls on the Data,,,Instrument Controls,,,,,,Detector,,,,FSC detector 250 gain,FSC detector 350 gain,,,,,,,,,,Review,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Data Processing,,,,,,,,,,,,,,Review Questions,Wh

12、ich of the following fluorochromes cannot be used with the FACSCalibur?DAPI (ex. 345 nm, emits 455 nm)Propidium Iodide (ex. 536 nm, emits 617 nm)Alexa Fluor 647 (ex. 650 nm, emits 668 nm)What are the three measureme

13、nts of a particle that can be determined by FACSCalibur?Briefly describe the functions of the fluidics, optics, and electronics systems.,Review Questions,What would happen to the population below if you increased the Re

14、d parameter value in the Instrument controls?,,Review Questions,Which instrument components ensure that the fluorescence signal of a specific fluorochrome is only measured by a designated detector? For example, APC is on

15、ly measured by the FL4 detector.,樣本處理,細(xì)胞懸液的制備,細(xì)胞懸液：分離PBMC、PRP等：操作復(fù)雜，分離、離心步驟導(dǎo)致細(xì)胞特定群體丟失，并可能引入某些誤差直接使用外周血、骨髓：最接近生理狀況，操作簡便，樣本用血量小灌洗液、體腔積液培養(yǎng)細(xì)胞、細(xì)胞系實體組織：病理組織：新鮮樣本/石蠟包埋樣本針吸組織：新鮮樣本鞘液、洗液等：清潔無顆粒雜質(zhì),步驟一： 選擇合適的熒光染料

16、,必須能夠被流式細(xì)胞儀上所配備的激光器所激發(fā)激發(fā)的光譜必須在儀器上濾光片能夠接受的合適范圍內(nèi)熒光素光譜的重疊應(yīng)當(dāng)盡量減少,熒光產(chǎn)生過程,,Propidium Iodide,,,,,,,,,,,,,,,,,400 nm,500 nm,600 nm,700 nm,,,,,PI,,,,,,,DNA,Excitation,Emisson,,300 nm 400 nm 500 nm 60

17、0 nm 700 nm,,Fluorescein (FITC),,,,,,,,,,,,,,,,,400 nm,500 nm,600 nm,700 nm,,,,,Wavelength,,,,,,,Protein,Excitation,Emisson,,300 nm 400 nm 500 nm 600 nm 700 nm,,Excitatio

18、n,Emisson,300 nm 400 nm 500 nm 600 nm 700 nm,Phycoerytherin (PE),,,,,,,,,,,,,,,,,,,,,,,,,,,Protein,,Allophycocyanin (APC),,,,,,,,,,,,,,,,,,,,,,,,,,,Protein,,,632.5 nm (HeNe),,Excita

19、tion,Emisson,300 nm 400 nm 500 nm 600 nm 700 nm,FITC和PE熒光光譜,補償調(diào)節(jié)前后對比,FL1,FL2,步驟二： 染色,體積溫度孵育時間對照,直接染色,,,,,,,,,Fluorescent probe attached to antibodySpecifi

20、c signal: weakNonspecific binding: low,間接染色,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Fluorescent probe attached to a 2nd antibodySpecific signal: strong, 5-6 2nd Ab/each 1st Ab;Nonspecific binding: high,Avidin-Biotin method I,

21、,,,,,,,,,,,,,,,,,,,,,,,biotinylated primary Ab,biotin,avidin,biotinylated dye,步驟三： 數(shù)據(jù)收集和分析,畫圖尋找目標(biāo)細(xì)胞調(diào)整儀器設(shè)置到合適的狀態(tài)我們可以得到怎樣的結(jié)果？它們意味著什么？,儀器設(shè)置調(diào)節(jié),1. 用未染色細(xì)胞調(diào)整儀器PMT電壓,2. 用單染色的細(xì)胞調(diào)節(jié)儀器補償,關(guān)于同型對照,例：一個雙色染色的實驗 抗體A

22、 FITC，抗體B PE 對應(yīng)的同型對照是IgG1 FITC,IgG1 PE那么需要準(zhǔn)備的是陰性對照：細(xì)胞加上IgG1 FITC,IgG1 PE單陽性對照： FITC: 細(xì)胞加上Ab A FITC, IgG1 PE PE: 細(xì)胞加上Ab B PE, IgG1 FITC,數(shù)據(jù)分析,設(shè)門設(shè)定陰性與陽性群體的界限確定陽性與陰性細(xì)胞群體統(tǒng)計陽性或陰性細(xì)胞群體的百分率，平均熒光值，絕對數(shù)

23、或抗體結(jié)合數(shù),如何設(shè)定陰性與陽性的界限,,,,,,細(xì)胞結(jié)構(gòu)細(xì)胞大小細(xì)胞粒度細(xì)胞表面面積核漿比例DNA含量與細(xì)胞周期RNA含量蛋白質(zhì)含量染色體分析,細(xì)胞功能細(xì)胞表面/胞漿/核的特異性抗原細(xì)胞活性細(xì)胞內(nèi)細(xì)胞因子酶活性激素結(jié)合位點細(xì)胞受體細(xì)胞凋亡,在上述信號基礎(chǔ)上的細(xì)胞分選,流式細(xì)胞術(shù)的細(xì)胞學(xué)應(yīng)用,流式細(xì)胞儀的臨床應(yīng)用,HIV免疫分型，CD4絕對計數(shù)白血病和淋巴瘤的免疫分型腫瘤的細(xì)胞周期和倍體分析網(wǎng)織紅細(xì)胞

24、計數(shù)細(xì)胞移植的交叉配型和免疫狀態(tài)監(jiān)測干細(xì)胞計數(shù)殘量白血病細(xì)胞檢查HLA-B27檢查血小板功能及相關(guān)疾病,流式細(xì)胞儀的科研應(yīng)用,免疫功能研究癌癥病人的多藥耐藥性細(xì)胞動力學(xué)功能研究動物性別篩選海洋與環(huán)境微生物分析染色體分選,流式細(xì)胞儀的遺傳學(xué)應(yīng)用,細(xì)胞DNA, RNA 含量的測定染色體倍體分析染色體分離基因表達(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)基因表達(dá)調(diào)控研究細(xì)胞內(nèi)基因定位酵母轉(zhuǎn)基因株的篩選報告基因

25、的定性定量檢測植物遺傳學(xué)研究 ??????,What can Flow Cytometer tell us about a cell?,細(xì)胞增殖（細(xì)胞周期）,細(xì)胞內(nèi)DNA含量增殖相關(guān)基因的表達(dá)BrdU的結(jié)合示蹤染料,DNA 探針,DNA探針最主要的特點是，它們與DNA含量是成化學(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Propi

26、dium IodideEthidium Bromide苯甲亞胺Hoechst 33342抗生素Mithramycin, Chromamycin A3Acridine Orange - AOPyronyn Y,,,,,,,,,,,,G2,M,G0,G1,s,0,200,400,600,800,1000,,,,,,,,,,,,,,,,,,,,,,s,DNA Analysis,DNA content,Count,Normal

27、Cell Cycle,A typical DNA Histogram,,,,,G0-G1,S,G2-M,Fluorescence Intensity,# of Events,紅色為正常二倍體細(xì)胞；黃色為異倍體細(xì)胞,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 antige

28、nKi-S1 - proliferation related antigenPhospho-histoneCyclin D1, E, A, B1,BrdUrd Incorporation,Bromodeoxyuridine (BrdU) 是一個胸腺核苷的類似物 能夠在細(xì)胞增殖和細(xì)胞周期狀態(tài)分析中起作用BrdU能與正在復(fù)制周期內(nèi)的細(xì)胞相結(jié)合使用BrdU抗體能夠檢測到BrdU,BrdUrd Incorporation,細(xì)胞分裂的

29、分析,哺乳動物的免疫系統(tǒng)需要有大量的增殖，能夠確?？乖禺愋缘腡細(xì)胞和B細(xì)胞具有足夠的增殖速度，能夠?qū)Ω吨尾∩镌斐傻母腥綜FSE是一種示蹤染料，能夠均等地在兩個子代細(xì)胞中分布，其結(jié)果可以區(qū)分出8-10代的子細(xì)胞。,Tracing Dye(CFSE),Apoptotic Cell Death -- a genetically encoded cell death program, which is morphologica

30、lly, biochemically and molecularly distinct from necrosis,Flow cytometry of apoptotic cell death,細(xì)胞散射光 在細(xì)胞調(diào)亡中，細(xì)胞收縮，從而FSC下降，SSC上升或是沒有明顯變化,Flow cytometry of apoptotic cell death,熒光吸收細(xì)胞的胞膜對于DNA染料的通透性和細(xì)胞的活性、死亡和凋亡相關(guān)，像PI、EB

31、、Hoechst-33342等，可以用來區(qū)分活細(xì)胞、死細(xì)胞和凋亡細(xì)胞,Flow cytometry of apoptotic cell death,FCM of Caspases通過抗體和活化的caspase-3片斷相結(jié)合來檢測 使用特異性的caspase-3熒光底物新的抗原決定基CK18,Flow cytometry of apoptotic cell death,線粒體功能的變化 TMP會在調(diào)亡中產(chǎn)生，可以通過一些標(biāo)記用流式

32、細(xì)胞儀檢測。使用有膜穿透性的親脂性陽離子熒光染料，如Rh123, DiOC6, JC-1,CMXRos等，可作為流式檢測的探針。 當(dāng)細(xì)胞發(fā)生調(diào)亡時，一個線粒體膜表面蛋白——7A6抗原會出現(xiàn)Bcl-2/bax family of proteins.,Flow cytometry of apoptotic cell death,鈣離子流和 pH值變化 胞漿內(nèi)Ca2+ 水平的上升和由于細(xì)胞內(nèi)環(huán)境酸化造成的選擇性的pH值的調(diào)節(jié)降低

33、是伴隨著細(xì)胞凋亡產(chǎn)生的后果之一。使用Ca2+ 選擇性熒光探針，如 Quin-2, Fluo-3, Indo-1 通過流式檢測是測定細(xì)胞內(nèi)Ca2+濃度的最佳方案酸化的檢測同樣可以用對pH敏感的熒光探針，如 DCH, BCECF, BCECF-AM, SNAFLs, SNARFs等進(jìn)行檢測,Flow cytometry of apoptotic cell death Phospholipid redistributi

34、on,Flow cytometry of apoptotic cell death,DNA 鏈的斷裂細(xì)胞凋亡晚期中，核酸內(nèi)切酶（某些Caspase的底物）在核小體之間剪切核DNA，產(chǎn)生大量長度在180-200 bp 的DNA片段。 斷裂的末端可以通過末端轉(zhuǎn)脫氧核苷酰酶(TdT)連接上dUTP。,Flow cytometry of apoptotic cell death,細(xì)胞DNA含量 在凋亡細(xì)胞中，用PI染色，通過流式檢測DNA含

35、量，可以發(fā)現(xiàn)一種亞群的細(xì)胞，其染色熒光強度下降。這是由于隨著調(diào)亡的進(jìn)行，核酸內(nèi)切酶活化，隨后一部分DNA泄漏出去，造成細(xì)胞內(nèi)DNA含量下降造成的。,Flow Cytometry of Apoptotic Cells,An Integrated Approach to Cell Immunology,Immune Function assay,免疫細(xì)胞亞群檢測Antigen-peptide specific T cells

36、檢測T-cells 活化Treg Cells細(xì)胞內(nèi)細(xì)胞因子/趨化因子檢測磷酸化,淋巴細(xì)胞亞群分析,根據(jù)功能，淋巴細(xì)胞主要分為B淋巴細(xì)胞（CD19+），與體液免疫有關(guān)T淋巴細(xì)胞（CD3+），與細(xì)胞免疫有關(guān)總T和總B可以用來判斷某些免疫缺陷和自身免疫性疾病 NK細(xì)胞（CD3-CD16+56+），行使免疫監(jiān)控功能， 能夠介導(dǎo)對某些腫瘤細(xì)胞和病毒感染細(xì)胞的細(xì)胞毒性作用。,淋巴細(xì)胞亞群分析,根據(jù)CD4、CD8表達(dá)，T淋巴細(xì)胞又分為

37、T輔助/誘導(dǎo)細(xì)胞（CD3+CD4+）T抑制/細(xì)胞毒性細(xì)胞（CD3+CD8+）Th/Ts 評價那些自身免疫失調(diào)或被懷疑是免疫失調(diào)或已知患有免疫缺陷的病人的免疫狀態(tài)，此外，這一比值還可用來監(jiān)測骨髓移植病人以免受到急性GVHD的攻擊 Th/Ts升高：自身免疫性疾?。愶L(fēng)濕性關(guān)節(jié)炎、SLE） Th/Ts降低：病毒感染、惡性腫瘤、再生障礙性貧血,Absolute Counts -- Cells per mL,Positive

38、 Cell,Absolute Count Beads,Negative Cell,CD4 PE,CD4 PE,抗原特異性T細(xì)胞,提供檢測者一個強有力的工具用于研究對病毒抗原的免疫應(yīng)答和疫苗的開發(fā),MHC: Class I and Class II Gene Products,T cell受體不直接和可溶性抗原相連抗原必須通過MHC由抗原呈遞細(xì)胞傳遞給T細(xì)胞 (Macrophages

39、, Dendritic cells, B cells)MHC Class I Gene Products呈遞抗原給 CD8+ T cells 誘導(dǎo)細(xì)胞毒應(yīng)答MHC Class II Gene Products呈遞抗原給 CD4+ T cells 誘導(dǎo)細(xì)胞因子產(chǎn)生，免疫球蛋白的分泌,MHC/TCR Signaling,二聚體可溶性MHC類似物與四聚體的比較,,Quantitation of antigen-specific

40、T lymphocytes in peripheral blood,HAM,Control,HIV,,CD8,Tax-A2/Ig,Gag-A2/Ig,Treg細(xì)胞的檢測,Treg細(xì)胞與自身免疫耐受相關(guān)，通常的檢測方法用CD4陽性細(xì)胞中CD25高表達(dá)，同時結(jié)合胞內(nèi)FoxP3含量來判斷最新發(fā)現(xiàn)人的Treg細(xì)胞低表達(dá)CD127。CD127的表達(dá)模式與Foxp3很接近，說明低表達(dá)CD127，高/中表達(dá)CD25的CD4陽性的T淋巴細(xì)胞就是Tr

41、eg細(xì)胞（調(diào)節(jié)性T細(xì)胞）。該方法比胞內(nèi)檢測Foxp3的優(yōu)點在于，檢測后的細(xì)胞可用于后續(xù)的培養(yǎng)及其他的體外試驗。另外的優(yōu)點是用這種檢測方案分選的Treg細(xì)胞得率更高。,,Immune Function Assays -- A Powerful Research Tool for Answering Basic Biological Questions in...,AIDS或癌癥機理的研究T cell亞群對于病毒和細(xì)菌抗原的反應(yīng)細(xì)胞介

42、導(dǎo)的對機會感染的免疫反應(yīng)藥物/疫苗的效果評價 免疫調(diào)節(jié)移植監(jiān)控毒理研究,Traditional Cytokine Flow Cytometry,IL-2 Phycoerythrin,CD4 FITC,細(xì)胞因子檢測,細(xì)胞因子是可溶性蛋白，在淋巴細(xì)胞免疫功能調(diào)節(jié)方面發(fā)揮重要作用。細(xì)胞因子可以調(diào)節(jié)多種細(xì)胞的生長、分化和功能，調(diào)節(jié)正常與病理狀態(tài)下的免疫應(yīng)答，研究生理或病理免疫調(diào)節(jié)因素所致細(xì)胞因子合成的應(yīng)答與改變是研究疾病病因與免疫狀

43、態(tài)的重要工具。,T細(xì)胞的細(xì)胞因子分型,Ⅰ型細(xì)胞介導(dǎo)免疫反應(yīng)引起遲發(fā)過敏反應(yīng)、巨噬細(xì)胞活化、2型反應(yīng)下調(diào)刺激來源：病毒、某些細(xì)菌Ⅱ型體液介導(dǎo)免疫反應(yīng)引起B(yǎng)細(xì)胞增殖、分泌多克隆免疫球蛋白、1型反應(yīng)下調(diào)刺激來源：多細(xì)胞寄生蟲、過敏源,細(xì)胞因子,Biological Variation Among CMV-seropositive Donors in Response to CMV,CFC & Proliferation,使

44、用溫和的方法進(jìn)行細(xì)胞破膜和固定，在中性pH值條件下，使細(xì)胞和BrdU結(jié)合，可以同時檢測到：S期 細(xì)胞周期 表型 活化狀態(tài) 核型決定基 胞漿內(nèi)決定基細(xì)胞因子/趨化因子其他…………..,CFC & Proliferation,Allows the correlation of: Phenotype Cytokine expressionCell CycleProliferation,BD Phosflow磷酸

45、化檢測系統(tǒng),更好的特異性的抗體建立在單個細(xì)胞基礎(chǔ)上的檢測可以同時檢測多個參數(shù)快速、靈敏、樣本量更少,P,,,,,P,,,,,,,,,,Y,Y,Y,Y,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

46、差異,BD PhosFLOW和Western blot 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 copies per cell. Sample 2 and 3 look the same via Western bl

47、ot, 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) 206–221),BD PhosFlow,Genomics & Prot

48、eomics -- Single Cells Have 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

49、 and pathways simultaniously.,CD20 PerCP-Cy5.5,CD3 PE,,,Zap70 (Y319)/Syk (Y352) Alexa 647,CD3 PE,,,CD3-/CD20+,CD3+/CD20-,CD3-/CD20-,CD20 PerCP-Cy5.5,Whole Blood CD3 CrossLink,Multi-Color PhosFlow in CD3/CD28

50、 or CD3 crosslinked human PBMCs or Whole Blood,CD3/CD28 Crosslink,,,Untreated cells (unshaded) vs treated cells (shaded),BD CBA,一種“三明治”免疫測定法—— 使用結(jié)合有高親和性抗體的小球，用來特異性地捕獲可溶性的抗原. 用熒光檢測抗體來檢測被捕獲的抗原 使用流式細(xì)胞

51、儀進(jìn)行檢測分析,Multiplexed Beads,,,,,,,,,,,,,,,,Shades of a color,Antibody coupled beads, emitting at distinct FL3 intensities,,,,Various analytes,,Antibody coupled PE label, emitting at FL2 intensity proportional to analyte co

52、nc.,,,,Multiplexed Beads,0 pg/mL,80 pg/mL,1250 pg/mL,5000 pg/mL,FL3 Beads,,FL2 (PE),,使用 BD? Cytometric Bead Array(CBA) 系統(tǒng)你可以:,? 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

53、? 如果是用488nm和635nm兩根激光器來實驗，實驗條件更容易建立? 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量檢測,目前CBA主要提供的檢測內(nèi)容,可溶性細(xì)胞因子細(xì)胞凋亡相關(guān)蛋白磷酸化蛋白,BD CBA Flex Set,,Old Format BD CBA Beads,BD CBA Flex Set Beads,更為靈活強大的CBA Flex Set,最多可同時檢測72個項目可自由選擇搭配檢測項目檢

54、測項目范圍更廣泛需要488nm和635nm兩種波長的激光器,9個指標(biāo)同時檢測活化T細(xì)胞,,1,2,3,4,5,6,7,8,9,1. Itk (Y511)2. ERK (T202/Y204)3. JNK (T183/Y185)4. P38 (T180/Y182)5. PLCg (Y783)6. ZAP70 (Y319)7. LAT (Y171)8. c-Jun (S63)9. RSK

55、(S380),Kinetics of Jurkat Cell Activation With Anti-CD3/CD28,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,1,10,100,FOLD INCREASE IN UNITS/ML,0,5,10,15,20,TIME (

56、MINUTES),,,,RSK (S380),,,Jun (S63),,,LAT (Y171),,,,Itk (Y511),,,,ZAP-70 (Y319),,,PLCg (Y783),,,,P38 (T180/Y182),,,JNK (T183/Y185),,,,ERK (T202/Y204),P-Specific Antibodies,Monitoring the T Cell Activation Pathway by CBA (

57、Control),RSK,,Cells were 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.,Monitoring the T Cell Activation Pathwa

58、y by CBA (PD-98059),Jurkat cells were pre-incubated with 200 mM PD-98059 (MEK inhibitor) for 20 minutes before being activated activated with anti-CD3/CD28 for 2 minutes. SDS was added to a final concentration of 1% and

59、the material was placed in a boiling water bath for 5 minutes.,Monitoring the T Cell Activation Pathway by CBA (PP2),Jurkat cells were pre-incubated with 10 mM PP2 (Src family kinase inhibitor) for 20 minutes before bein

60、g activated 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.,FCM in Molecular Biology,流式檢測分子表型特異的核酸序列SNP 端粒長度

61、熒光報告蛋白檢測基因表達(dá)情況,Fluorescent Proteins,DsRedZsYellowHcRedAmCyan,端粒長度的檢測,端粒的長度和端粒酶的活性是現(xiàn)在研究的重點。因為它們被發(fā)現(xiàn)和細(xì)胞復(fù)制以及一些疾病的變化相關(guān)?？梢酝ㄟ^FISH，用帶有熒光的核酸探針標(biāo)記上端粒序列，通過流式細(xì)胞儀檢測端粒的長度,流式分選應(yīng)用舉例,,自然殺傷細(xì)胞分選,,,,,基因轉(zhuǎn)染細(xì)胞的分選,綠色螢光蛋白分析（GFP）：左圖顯示Hela細(xì)胞轉(zhuǎn)