流式细胞仪工作原理

1、流式细胞仪工作原理流式细胞仪工作原理刘 益 年产品应用专员E mail: FACSCalibur大纲 实验设计原理 流式细胞仪运作原理 FACSCalibur 操作流程456AdhesionAdhesionReceptorsReceptorsMetabolicMetaboliccytokinescytokinesstructurestructureenzymesenzymes7Lysed Whole Blood Components8Example91011Mapping normal and cancer cell signalling networks: Mapping normal an

2、d cancer cell signalling networks: towards single-cell proteomics. towards single-cell proteomics. Nat Rev Cancer. 2006 Feb;6(2):146-55 Nat Rev Cancer. 2006 Feb;6(2):146-55 12Development Of Visualization ToolsNolan et al.13Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT, Nolan GP.

3、Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 2004 Jul 23;118(2):217-28.Clustering of Biosignature, Clinical Significance141516Annexin V PEAnnexin V PE0 0.5 1 0 0.5 1 2 42 4Incubation with Camptothecin (hr)Incubation with Camptothecin (hr)17PIPIAnnexin V-FITCAn

4、nexin V-FITC1819N+C2H5NH2NH2EthidiumN+C2H2)3NH2NH2N+C2H5CH3C2H5Propidium200 200 400 600 8001000DNA contentDNA contentCount2122232425262728Gervaix, A. et. al. 1997. A Gervaix, A. et. al. 1997. A new reporter cell line to new reporter cell line to monitor HIV infection and monitor HIV infection and dr

5、ug susceptibility in drug susceptibility in vitro. PNAS vol. 94.vitro. PNAS vol. 94.293031Cytometric Beads Array (CBA)Single StepIncubation Two-StepIncubation or or 32Multiple Multiple sizessizesBeads provide an expandable assay platform Beads provide an expandable assay platform for use with a flow

6、 cytometerfor use with a flow cytometerDifferent Different intensitiesintensities* *Different colors Different colors with different with different intensitiesintensities33Proteins Measured A. Interleukin (IL)-2 B. IL-4 C. IL-5 D. IL-10 E. Tumor Necrosis Factor-a F. Interferon-g 34Zap 70 Detection.3

7、5Kinetic analysis of T cell activation by anti-CD3/CD2836Philadelphia chromosome The presence of this translocation is a highly sensitive test for CML, since 95% of people with CML have this abnormality 37Philadelphia chromosome缓冲液液流区缓冲液液流区样品流动区侦测区激發光源-螢光螢光-散射光散射光 流式细胞仪的工作原理流式细胞仪能测量 : 散射光 细胞大小 (前方散射

8、光) 细胞折射率 (侧方散射光) 各色萤光 液流系统:将细胞依序送到测量区受检。 光学系统:产生并收集萤光、光散射等信号。 电子系统: 将光学讯号转换成电子讯号。 分析所输出的电流讯号，以脉冲高度、宽度、积分面积显示。 量化讯号并传至电脑。 综合三个系统的功能:综合三个系统的功能-液流系统FACS Calibur 的液流系统FACS Calibur 仪表板LO = 12uL/minMED = 35uL/minHI = 60uL/minPRIME= Drain & FillSTANDBYRUNred laserblue laserLow Sample PressureLow Sample

9、 PressureHigh Sample PressureHigh Sample Pressuresheathsheathsamplesheathsheathsample综合三个系统的功能-光学系统LONG (700nm)550 Long Pass (650LP)650 Short Pass (600SP)SHORT (500nm)Pass Through Filters600/100 Band Pass (600/100)Transmitted 550 nm550 - 650 nm (60050)Wavelengths Blocked650 nm550 nm650 nmShort PassL

10、ong PassBand PassLONG (700nm)550 Long Pass (650LP)650 Short Pass (600SP)SHORT (500nm)Dichroic FiltersTransmitted 550 nmDiflected 90650 nm550 nm萤光滤片FACS Calibur 的光学系统488 nm综合三个系统的功能-电子系统 将光学讯号转换成正比例的电子讯号。 分析所输出的电子讯号，以脉冲高度、宽度、积分面积显示。 将量化讯号传至电脑。 电子系统电子系统电位脉冲之定量电位脉冲之定量讯号之产生与处理Amp Gain 1.0-9.99The Use of

11、 Linear AmplificatonThe Use of Linear AmplificatonAssume we convert linear analog signals using a 10 bit ADC-we have 1024 channels of range corresponding to 0-10 V.Channel difference is 10V/1024= 0.01VIdeal Lin To Log ConversionIdeal Lin To Log Conversion实验数据的呈现15 to 20 mm Monocyte 10 to 14 mm Neutr

12、ophil8 to 10 mmLymphocyte散点图反映细胞形态 常见的数据呈现直方图分析报告 CV=S.D./Mean散点图分析报告 FACSCalibur-复习综合三个系统的功能仪器之调试 调整FSC / SSC 散点图以圈选目标细胞群 设阈参数及阈值 调整萤光信号接受器 FL 1-4 色差补偿 仪器之调试 调整FSC / SSC 散点图以圈选目标细胞群 设阈参数及阈值 调整萤光信号接受器 FL 1-4 色差补偿 散射光信号的调整散射光信号的调整散射光信号的调整Cell Line仪器之调试 调整FSC / SSC 散点图以圈选目标细胞群 设阈参数及阈值 调整萤光信号接受器 FL 1-4

13、 色差补偿 阈值的调整仪器之调试 设阈参数及阈值 调整FSC / SSC 散点图以圈选目标细胞群 调整萤光信号接受器 FL 1-4 色差补偿 自体萤光信号的调整 Single仪器之调试 设阈参数及阈值 调整FSC / SSC 散点图以圈选目标细胞群 调整萤光信号接受器 FL 1-4 色差补偿 FITCPE450500550600650Remember the basic assumption of flow analysis:Remember the basic assumption of flow analysis: The signal in FL1= the signal from FI

14、TC and only FITC and The signal in FL1= the signal from FITC and only FITC and the signal in FL2= the signal from PE and only PE. the signal in FL2= the signal from PE and only PE.This is NOT TRUE for the raw data! The process by which each fluorescence channel is “corrected for this spectral overla

15、p is termed Fluorescence CompensationFL1 = FITC + x% PEFL1 = FITC + x% PEFL2 = PE + y% FITCFL2 = PE + y% FITC萤光补偿调整 (The Problem)萤光补偿调节FL2 - %FL1FL-1(FITC)FL-2(PE)FL1 - %FL2(18 30%)(0 1%)萤光补偿调节(Compensation Fitc)萤光补偿调节(Compensation Pe1)萤光补偿调节(Compensation Pe2)萤光补偿调节(Compensation PerCP)FACSCalibur 各部

16、构造 检体吸取区检体吸取区(SIP)(SIP)Droplet Containment System包含：a. 外管b. 真空泵清除内管内前一个检体残留物c. 上样管(内管)內管(SIT)底座Bal sealFACSCaliburFACSCalibur仪表板仪表板LO = 12uL/minMED = 35uL/minHI = 60uL/minPRIME= Drain & FillSTANDBYRUN正确开机程序正确开机程序开启细胞仪电源。开启其他周边配备电源，如打印机及 M. O.。开启电脑。确认鞘流液筒有八分满的 FACS FLOW，旋紧。将废液倒掉，并在废液筒中加入100 c.c.

17、家用漂白水。将气压阀方向调在加压（Pressurize）位置。排除液流过滤器中的气泡。使用 1 c.c. PBS 为样品，执行 PRIME 功能两次。HIGH RUN 两分钟，即可开始分析样品。仪器清洗与关机正确步骤仪器清洗与关机正确步骤 清洗步骤要确实：尤其是使用PI之后Prime (Drain then Fill) 2X 3X ：冲洗 Flow cell 区FACS Rinse (或general detergent) 3 ml- 底座向右移，吸取约2ml：清洗外管- 底座移回中央，High Run 5 min：清洗内管內管(SIT)底座FACS Clean (或10% bleach) 同Step 2.Prime 2X 3XDistilled water 同Step 2.管内仅留 1 ml dH2OStandby for 5 min关机仪器清洁度确认设置取 1 ml FACSFlow（ 或确实纯净dH2O）上样以如下建议仪器条件跑样品设置条件1: Threshold=FL1 set 52, FL1 500 (Log) Amplification， HI RUN。在此情况下，Counter 窗口中event rate 应为 0/sec 接着以如下建议仪器条件跑样品设置条件2: Threshold=FSC set 52, FSC E0