流式细胞仪试剂介绍

流式细胞仪试剂介绍,五光十色的荧光素,单分子荧光素耦合的复合荧光素分子（Tandem Dye)新型的荧光素样荧光物质Qdots,常用激光和荧光素搭配,BlueFITCPEPerCPPerCP-Cy5.5PE-Texas Red(ECD)PE-Cy5PE-Cy7PIEGFP,RedAPCAPC-Cy7UVDAPIHoechst,2010,2005,2000,1995,1990,Innovation First Commercial Tandem Conjugates,1985,PE-Cy5.5,PE-Texas Red,PE-Cy7,PE-Cy5,APC Alexafluor700APC Alexafluor750,Multicolor Flow Impossible Without Tandems,Beckman Coulter - Patented Conjugation Process,Patented Process Unique To Beckman Coulter,Native State,Unfold,Couple Acceptor Dye,Refold,Performance,Standardization,Tandem Video,Unpurified PC7 Discarded Material Purified PC7,Performance Matters: Patented Manufacturing Process,Finished Tandem DyeThree Lot Comparison of PC7,Purification,Reproducible Data Over Time,Performance,Standardization,Performance Matters: Vendor Comparison,Clear Resolution of Cellular Sub-sets,Beckman Coulter,Performance,Vendor X,Vendor Y,Performance Matters: Impact Of Photo-degradation,Patented Packaging Protects Against Photo-degradation,Performance,荧光染料比较(平均荧光强度) Q-Prep / ImmunoPrep 试剂系统,PerCP APC FITC ECD PC5 25 reagent vials4 hoursRe-titrationRe-validation,PERFORMANCE,EFFICIENCY,Multicolor Panels Workflow,10 specimens with five color panelsFrom receipt to first results,Simplified Workflow, Standardized, Faster,PERFORMANCE,EFFICIENCY,7 reagent vials3 hoursPre-titratedPre-validated,A66286B-Cell KitA66287T-Cell KitA66288Myeloid Kit,Solastra 5-Color Panels Part Numbers *,* Not Available in the USA,EFFICIENCY,科研试剂,Cell SignalingAutomationiTAG Tetramer Technology,Cancer Research Focused On Signaling Pathways,Gleevec,Illustration courtesy of Dr David Hedley, Princess Margaret Hospital,200 of 208 New Drugs Are PKIs,Performance,Flexibility,Standardization,The Competition: Western Blot vs Flow Cytometry,Flow: Visualization of Signaling in Discrete Cell Populations,Lysates Homogeneous data Qualitative Slow,Flow Cytometry,Identifiable cells Multiple populations Quantitative Fast,Performance,Flexibility,Standardization,Date kindly suppled by Dr Vincent Shankey, BCI Miami, FL, USA,MAPK - AF488,Red = Un-stimulatedBlue = Stimulated,Challenges of Cell Signaling: Assay Standardization,Timing,Temp,Reagents,Multiple Opportunities For Variability in Results,Specimen,Operator,Performance,Flexibility,Standardization,Example of Optimization: Standardized ZAP-70 Assay,Maximized Signal to Noise Ratio With BCI Method,Optimized Sample Prep to Yield Best S/N Ratio,BCI,CD19,CD3,CD56,Company B,Company C,Company A,ZAP-70 PE,ZAP-70 PE,ZAP-70 PE,ZAP-70 PE,Performance,Standardization,S/N Ratio = ZAP-70 MFI (T-cells)/ZAP-70 MFI Normal B-cells,MHC Tetramer技术,MHC TETRAMER是4个MHC 分子的复合体, MHC分子分为I, II类分子. I类分子介导与CD8+细胞亚群反应. II类分子介导与CD4+细胞亚群反应. 常用的是I类分子. 每个MHC分子都结合一个生物素分子, 一个亲合素分子又和4个生物素分子结合, 这样,4个MHC分子就结合到一起.亲合素标记某一种荧光素,可以用流式细胞仪检测出来. MHC分子并不是直接与T细胞上的TCR结合,而是通过多肽做为桥梁结合在一起的.因此,不同的多肽结合的T细胞是不同的.简单的说, MHC TETRAMER技术可以检测细胞对某一种多肽的反应性.,How do we Differentiate Our Tetramers,The a3 mutation see next slideALL of our Tetramers are manufactured using GMP compliant procedures important for clinical trial customersQuality, Quality, Quality,3 Domain Binding ModificationComparison with unmodified, “native” Tetramers,B,优点,1.可以进行定量.2.不需要放射性同位素,无污染.3.快速, 可以处理大量的血液和组织样本.4.由于用流式分析, 可同时标记TETRAMER 和其他的表面标志,进行多参数的分析.5.无需破坏细胞, 得到的阳性细胞可通过流式分选下来,进行功能性的研究.6无需预先体外培养,即可分析特异的T细胞.,应用范围,感染(病毒, 寄生虫)癌症自身免疫病移植,BECKMAN COULTER公司的TETRAMER产品,iTAg MHC Tetramers,BECKMAN COULTER公司的TETRAMER产品,iTAg MHC Tetramers IFN-r kit 可以与iTAg MHC Tetramers 共同使用,同时定量特异性T细胞和IFN-r的产生,检测细胞的功能状态.,Combined Tetramer / IFN Assay,Figure A: CD8+ IFN-g+ cells. 3.53% is reported as antigen specific T cells not correct!Figure B: The true number of antigen specific T cells (Tetramer+ cells) is 1.91% + 1.07% = 2.98%Also in Figure B, the number of “bystander cells” is revealed in the bottom right hand quadrant. Since these cells are Tetramer-, they are NOT antigen specific T cells, but are non-specificallySecreting IFN-g,Competition: iTAg vs ProImmune Pentamers,iTAg PBMC Staining protocol,Pro5 PBMC Staining protocol,iTAg MHC TetramerHLA-A*0201 CMV pp65,Method B MHC PentamerHLA-A*0201 CMV pp65,iTAg Tetramers: When results literally count,Method B Cell staining protocol requires incubation on ice Method 2X longer (90 minutes vs. 40 minutes) Antibodies need separate incubation: more steps,iTAg MHC Tetramer Higher S/N ratio, excellent population separation Rapid procedure, faster time to result Fewer steps,Manual Intracellular Cytokines: Variability,Whole Blood 18hr SEB+CD28 30 tubes at one time: 19 replicates CD4/ IFNg /CD69,Gated on CD4+Lymphs:,Date kindly supplied by Dr Wade Bolton, Custom BioPharma Services, Miami, FL, USA,Automated Intracellular Cytokines: Precision,Whole Blood 18hr SEB+CD28 30 replicates at once CD4/ IFNg /CD69,Gated on CD4+Lymphs:,Date kindly supplied by Dr Wade Bolton, Custom BioPh