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1、23-13536-00 Rev.01,Fluorochromes,23-13536-00 Rev.01,2,Fluorochrome Properties,Desirable properties for fluorochromes:High relative brightnessNarrow emission spectrum(low spectral overlap in combination)Easily conjugated(for immunophenotyping)Fluorochromes can be characterized by:Type of moleculeExci
2、tation and emission wavelengthsRelative brightness,23-13536-00 Rev.01,3,Fluorochrome Molecule Types,Small organic moleculesexamples:FITC,BD HorizonTM V450,Cy7Fluorescent proteinsexamples:PE,APC,PerCPTandem dyestypically,the coupling of a fluorescent protein donor with a small organic molecule accept
3、orexamples:PE-Cy7,PerCP-CyTM5.5Nanocrystals(Qdots)inorganic semiconductorsexamples:Qdot 565,Qdot 605,23-13536-00 Rev.01,4,Some Common Fluorochromes,23-13536-00 Rev.01,5,Small Organic Fluorochromes,AdvantagesLow molecular weightEasy to conjugatedirect attachment to free amino groups on mAbExcellent s
4、tabilityExtremely consistent emission spectraDisadvantagesSmall Stokes Shift(50100 nm)Tend to be less bright,23-13536-00 Rev.01,6,Protein Fluorochromes,AdvantagesGood stabilityConsistent emission spectraMedium Stokes Shift(75200 nm)Tend to be more brightDisadvantagesHigh molecular weightMore difficu
5、lt to conjugateintermediaries needed to attach to mAb,23-13536-00 Rev.01,7,Tandem Dye Fluorochromes,AdvantagesVery large Stokes Shift(150300 nm)Tend to be very brightoften brighter than the fluorescent protein donor DisadvantagesHigh molecular weight(similar to fluorescent protein)Difficult to make
6、consistently(lot-to-lot variation in emission properties)Harder to conjugate(same as fluorescent protein)Some tandems have poor stability,23-13536-00 Rev.01,8,Nanocrystal Fluorochromes,AdvantagesLarge Stokes Shift(100500 nm)Tend to be very brightEmission peaks are consistent and narrow,and do not ch
7、ange with variations in the excitation sourceHighly resistant to photobleachingNanocrystals share biophysical and conjugation properties DisadvantagesDifficult to conjugateInstability of bindingsCytotoxicityWide excitation range produces cross-laser spillover,23-13536-00 Rev.01,9,Excitation and Emis
8、sion,Excitation wavelengths determine lasers that can excite the fluorochrome.Emission wavelengths determine filters and PMTs that can measure the emission signal.,23-13536-00 Rev.01,10,Know Your Cytometer,Cytometer ConfigurationBD FACSCantoTM II 4-2-2 configuration is shown below BDTM LSR II 4-2-2
9、configuration is similar,4 detectors for blue laser,2 detectors for red laser,2 detectors for violet laser,23-13536-00 Rev.01,11,Typical Excitation and EmissionBD FACSCanto II 4-2-2(BD LSR II 4-2-2 is similar),23-13536-00 Rev.01,12,Fluorochrome Use Depends on the Cytometer Configuration,23-13536-00
10、Rev.01,13,Fluorochrome Brightness,The brightness of a fluorochrome depends on two factors:Molar Extinction Coefficient()measures how well a fluorochrome absorbs energy.Quantum Yield(Qy)is the ratio of photons emitted to photons absorbed.Brightness=x QyRelative Brightness=,Brightness of PE,Brightness
11、,23-13536-00 Rev.01,14,Some Fluorochromes are MUCH Brighter,PE is 50 x brighter than FITC and 10 x brighter than APC.APC is 5x brighter than Pacific Blue.Extinction coefficient is more significant than quantum yield in determining brightness.,23-13536-00 Rev.01,15,D,D=difference between the medians
12、of the positive and negative populationsW=spread(2 x rSD)of the negative population,Stain Index,Stain Index=,Stain index is a practical way to characterize the brightness of a marker with respect to a given optical configuration.,DW,W,Stain index can also be used to characterize the sensitivity of a
13、 fluoresence parameter.,23-13536-00 Rev.01,16,Typical CD4 Stain IndexesBD LSR II,APC has a higher CD4 stain index than PE-Cy5,but approximately 1/10th the relative brightness.,23-13536-00 Rev.01,17,Typical CD4 Stain IndexesBD FACSCanto II,FITC has approximately the same CD4 stain index as PerCP,but
14、approximately 1/10th the relative brightness.,23-13536-00 Rev.01,18,Stain Index Factors,Stain index is dependent on the optical configuration and additional performance factors.Factors that can affect stain index include:Laser wavelength and powerDetector rangeDetector efficiencyBackground signalDon
15、t depend on published valuesmeasure stain index on your own system.,23-13536-00 Rev.01,19,CD4 Stain Indexes Across Cytometers,Stain Index Exercise,23-13536-00 Rev.01,20,Fluorescence Spillover,Emission of FITC in PE channel,23-13536-00 Rev.01,21,Significant Spillovers on 4-2-2 Configuration,23-13536-
16、00 Rev.01,22,Spillover Decreases Sensitivity,Without CD45 AmCyan,With CD45 AmCyan,CD19 FITC,Spillover can significantly increase the variability of negative and dim populations,even after compensation is applied.,23-13536-00 Rev.01,23,Lost Population due to Spillover,Lymphocytes stained with CD45 FI
17、TC and CD4 PE,CD45 FITC causes dim CD4+CD45+to be difficult to distinguish due to significant FITC spillover into PE.,Lymphocytes stained with CD45 PerCP and CD4 PE,CD45 PerCP allows dim CD4+CD45+to be distinguished from backgrounddue to minimal PerCP spillover into PE.,23-13536-00 Rev.01,24,Tandem
18、Dye Issues,Use tandem dyes in research applications with consideration of their technical limitations.APC-Cy7(and to a lesser extent PE-Cy7)can degrade in the presence of light,fixation,and elevated temperature.Degradation causes lower emission in the Cy7 detector and higher emission in the detector
19、 of the parent dye(APC or PE).APC-H7,APC-Cy7,and PE-Cy7 performance can be affected by polystyrene tubes.,23-13536-00 Rev.01,25,Tandem DegradationFalse Positives,False positives in APC channel reduced in absence of APC-Cy7,With CD8 APC-Cy7,WithoutCD8 APC-Cy7,23-13536-00 Rev.01,26,Tandem Degradation
20、Over Time,0 hours,2 hours,20 hours,PE,CD3 PE-Cy5,CD8 PE-Cy7,Time Sample Left in Light,PE,PE,23-13536-00 Rev.01,27,APC-H7 Is More Stable Than APC-Cy7,Comparison of Sample Stability,(in BD Stabilizing Fixative at RT),0,50,100,150,200,250,0,1,2,4,6,8,24,48,Hours of light exposure,%Spillover,23-13536-00
21、 Rev.01,28,Tandem Dye Recommendations,When using APC-Cy7 and PE-Cy7,beware of fixative and light instability issues.If problems arise when using polystyrene with APC-H7,APC-Cy7,or PE-Cy7,try switching to K-resin or polycarbonate.PerCP-Cy5.5 is less susceptible to instability than APC-Cy7 and PE-Cy7.
22、BD offers APC-H7 conjugated antibodies that are more stable than APC-Cy7 conjugates and have less spillover into the APC detector.,23-13536-00 Rev.01,29,New Violet Laser Fluorochromes,BD Horizon V450Maximum excitation at 404 nm,emission peak of 448 nm.BDHorizon V450 reagents exhibit performance comp
23、arable to Pacific Blue reagents as measured by Stain Index.Improved stability with fixation compared to Pacific Blue.,23-13536-00 Rev.01,30,New Violet Laser Fluorochromes,BD Horizon V500Maximum excitation at 415 nm,emission peak of 500 nm.Provides significantly reduced spillover into the FITC channe
24、l compared to AmCyanV500 has low excitation with the blue laser.Improved stability with fixation compared to AmCyan.,23-13536-00 Rev.01,31,BD Horizon V500 Low Spillover into FITC,V500 stained cells,AmCyan stained cells,Data is uncompensated.,23-13536-00 Rev.01,32,Factors Affecting Reagent Performanc
25、e,Relative brightness of the fluorochromeNumber of fluorochromes per antibodyExpression levels on(or in)the cells of interestBackground contributionsSpilloverPhotostabilityReagent environmentCytometer configuration,23-13536-00 Rev.01,33,Non-Conjugated Fluorescent Dyes,These dyes bind directly to cel
26、l components.Viability dyes discriminate live cells from dead cells.examples:7-AAD,TO-PRO-3,PI,DAPI,and the LIVE/DEAD series.Proliferation dyes are used to study cell division.examples:CSFE,PKH 67,and BD Horizon VPD 450.DNA dyes are used for DNA cell-cycle anaylsis.examples:PI,DAPI,and Hoechst 33342
27、.Reporter dyes are used to study gene expression.examples:mCherry and GFP.,23-13536-00 Rev.01,34,Non-Conjugated Fluorescence Dye DetectionBD FACSCanto II 4-2-2 and BD LSR II 4-2-2,mCherry is best detected with a 561-nm laser option and a 590630 detector range.,23-13536-00 Rev.01,35,BD Fluorescence Spectrum Viewer,the Fluorescence Spectrum Viewer to help choose fluorochromes based on:the number of colors you need in your experimentyour cytometer configurationspillover issues,
