《流式细胞技术荧光抗体课件.ppt》由会员分享，可在线阅读，更多相关《流式细胞技术荧光抗体课件.ppt（35页珍藏版）》请在三一办公上搜索。
1、Fluorochromes,23-13536-00 Rev. 01,Fluorochromes23-13536-00 Rev.,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
2、moleculeExcitation and emission wavelengthsRelative brightness,2,23-13536-00 Rev. 01,Fluorochrome PropertiesDesirab,Fluorochrome Molecule Types,Small organic moleculesexamples: FITC, BD HorizonTM V450, Cy7Fluorescent proteinsexamples: PE, APC, PerCPTandem dyestypically, the coupling of a fluorescent
3、 protein donor with a small organic molecule acceptorexamples: PE-Cy7, PerCP-CyTM5.5Nanocrystals (Qdots)inorganic semiconductorsexamples: Qdot 565, Qdot 605,3,23-13536-00 Rev. 01,Fluorochrome Molecule TypesSma,Some Common Fluorochromes,4,23-13536-00 Rev. 01,Some Common FluorochromesFluor,Small Organ
4、ic Fluorochromes,AdvantagesLow molecular weightEasy to conjugatedirect attachment to free amino groups on mAbExcellent stabilityExtremely consistent emission spectraDisadvantagesSmall Stokes Shift (50100 nm)Tend to be less bright,5,23-13536-00 Rev. 01,Small Organic FluorochromesAdv,Protein Fluorochr
5、omes,AdvantagesGood stabilityConsistent emission spectraMedium Stokes Shift (75200 nm)Tend to be more brightDisadvantagesHigh molecular weightMore difficult to conjugateintermediaries needed to attach to mAb,6,23-13536-00 Rev. 01,Protein FluorochromesAdvantage,Tandem Dye Fluorochromes,AdvantagesVery
6、 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 consistently (lot-to-lot variation in emission properties)Harder to conjugate (same as fluorescent protein)Some
7、tandems have poor stability,7,23-13536-00 Rev. 01,Tandem Dye FluorochromesAdvant,Nanocrystal Fluorochromes,AdvantagesLarge Stokes Shift (100500 nm)Tend to be very brightEmission peaks are consistent and narrow, and do not change with variations in the excitation sourceHighly resistant to photobleach
8、ingNanocrystals share biophysical and conjugation properties DisadvantagesDifficult to conjugateInstability of bindingsCytotoxicityWide excitation range produces cross-laser spillover,8,23-13536-00 Rev. 01,Nanocrystal FluorochromesAdvan,Excitation and Emission,Excitation wavelengths determine lasers
9、 that can excite the fluorochrome.Emission wavelengths determine filters and PMTs that can measure the emission signal.,9,23-13536-00 Rev. 01,Excitation and EmissionExcitat,Know Your Cytometer,Cytometer ConfigurationBD FACSCantoTM II 4-2-2 configuration is shown below BDTM LSR II 4-2-2 configuration
10、 is similar,4 detectors for blue laser,2 detectors for red laser,2 detectors for violet laser,10,23-13536-00 Rev. 01,Know Your CytometerCytometer C,Typical Excitation and EmissionBD FACSCanto II 4-2-2 (BD LSR II 4-2-2 is similar),11,23-13536-00 Rev. 01,Typical Excitation and Emissio,Fluorochrome Use
11、 Depends on the Cytometer Configuration,12,23-13536-00 Rev. 01,Fluorochrome Use Depends on t,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 emit
12、ted to photons absorbed.Brightness = x QyRelative Brightness =,Brightness of PE,Brightness,13,23-13536-00 Rev. 01,Fluorochrome BrightnessThe bri,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 i
13、s more significant than quantum yield in determining brightness.,14,23-13536-00 Rev. 01,Some Fluorochromes are MUCH Br,D,D = difference between the medians of the positive and negative populationsW = spread (2 x rSD) of the negative population,Stain Index,Stain Index =,Stain index is a practical way
14、 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 fluoresence parameter.,15,23-13536-00 Rev. 01,DD = difference between the me,Typical CD4 Stain IndexesBD LSR II,APC has a higher CD4 stain
15、 index than PE-Cy5, but approximately 1/10th the relative brightness.,16,23-13536-00 Rev. 01,Typical CD4 Stain IndexesBD L,Typical CD4 Stain IndexesBD FACSCanto II,FITC has approximately the same CD4 stain index as PerCP, but approximately 1/10th the relative brightness.,17,23-13536-00 Rev. 01,Typic
16、al CD4 Stain IndexesBD F,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 signalDont depend on published valuesmeasure stain i
17、ndex on your own system.,18,23-13536-00 Rev. 01,Stain Index Factors Stain inde,CD4 Stain Indexes Across Cytometers,Stain Index Exercise,19,23-13536-00 Rev. 01,CD4 Stain Indexes Across Cytom,Fluorescence Spillover,Emission of FITC in PE channel,20,23-13536-00 Rev. 01,Fluorescence SpilloverEmission,Si
18、gnificant Spillovers on 4-2-2 Configuration,21,23-13536-00 Rev. 01,Significant Spillovers on 4-2-,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.,2
19、2,23-13536-00 Rev. 01,Spillover Decreases Sensitivit,Lost Population due to Spillover,Lymphocytes stained with CD45 FITC 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 allow
20、s dim CD4+CD45+to be distinguished from backgrounddue to minimal PerCP spillover into PE.,23,23-13536-00 Rev. 01,Lost Population due to Spillov,Tandem Dye Issues,Use tandem dyes in research applications with consideration of their technical limitations.APC-Cy7 (and to a lesser extent PE-Cy7) can deg
21、rade in the presence of light, fixation, and elevated temperature.Degradation causes lower emission in the Cy7 detector and higher emission in the detector of the parent dye (APC or PE).APC-H7, APC-Cy7, and PE-Cy7 performance can be affected by polystyrene tubes.,24,23-13536-00 Rev. 01,Tandem Dye Is
22、suesUse tandem dy,Tandem DegradationFalse Positives,False positives in APC channel reduced in absence of APC-Cy7,With CD8 APC-Cy7,WithoutCD8 APC-Cy7,25,23-13536-00 Rev. 01,Tandem DegradationFalse Posit,Tandem Degradation Over Time,0 hours,2 hours,20 hours,PE,CD3 PE-Cy5,CD8 PE-Cy7,Time Sample Left in
23、 Light,PE,PE,26,23-13536-00 Rev. 01,Tandem Degradation Over Time0,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,27,23-13536-00 Rev. 01,APC-H7 Is More Stable Than APC,Tand
24、em 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.BD offers
25、APC-H7 conjugated antibodies that are more stable than APC-Cy7 conjugates and have less spillover into the APC detector.,28,23-13536-00 Rev. 01,Tandem Dye RecommendationsWhen,New Violet Laser Fluorochromes,BD Horizon V450Maximum excitation at 404 nm, emission peak of 448 nm.BDHorizon V450 reagents e
26、xhibit performance comparable to Pacific Blue reagents as measured by Stain Index.Improved stability with fixation compared to Pacific Blue.,29,23-13536-00 Rev. 01,New Violet Laser Fluorochromes,New Violet Laser Fluorochromes,BD Horizon V500Maximum excitation at 415 nm, emission peak of 500 nm.Provi
27、des significantly reduced spillover into the FITC channel compared to AmCyanV500 has low excitation with the blue laser.Improved stability with fixation compared to AmCyan.,30,23-13536-00 Rev. 01,New Violet Laser Fluorochromes,BD Horizon V500 Low Spillover into FITC,V500 stained cells,AmCyan stained
28、 cells,Data is uncompensated.,31,23-13536-00 Rev. 01,BD Horizon V500 Low Spillover,Factors Affecting Reagent Performance,Relative brightness of the fluorochromeNumber of fluorochromes per antibodyExpression levels on (or in) the cells of interestBackground contributionsSpilloverPhotostabilityReagent
29、 environmentCytometer configuration,32,23-13536-00 Rev. 01,Factors Affecting Reagent Perf,Non-Conjugated Fluorescent Dyes,These dyes bind directly to cell components.Viability dyes discriminate live cells from dead cells. examples: 7-AAD, TO-PRO-3, PI, DAPI, and the LIVE/DEAD series.Proliferation dy
30、es 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.Reporter dyes are used to study gene expression. examples: mCherry and GFP.,33,23-13536-00 Rev. 01,Non-Conjugated Fluorescent Dye,No
31、n-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.,34,23-13536-00 Rev. 01,Non-Conjugated Fluorescence Dy,BD Fluorescence Spectrum Viewer,bdbiosciences/spectraUse the Fluorescence Spectrum Viewer to help choose fluorochromes based on:the number of colors you need in your experimentyour cytometer configurationspillover issues,35,23-13536-00 Rev. 01,BD Fluorescence Spectrum Viewe,
