流式細胞技術(shù)熒光抗體課件

1、Fluorochromes23-13536-00 Rev. 01Fluorochromes23-13536-00 Rev. Fluorochrome PropertiesDesirable properties for fluorochromes:High relative brightnessNarrow emission spectrum (low spectral overlap in combination)Easily conjugated (for immunophenotyping)Fluorochromes can be characterized by:Type of mol

2、eculeExcitation and emission wavelengthsRelative brightness223-13536-00 Rev. 01Fluorochrome PropertiesDesirabFluorochrome Molecule TypesSmall organic moleculesexamples: FITC, BD HorizonTM V450, Cy7Fluorescent proteinsexamples: PE, APC, PerCPTandem dyestypically, the coupling of a fluorescent protein

3、 donor with a small organic molecule acceptorexamples: PE-Cy7, PerCP-CyTM5.5Nanocrystals (Qdots)inorganic semiconductorsexamples: Qdot 565, Qdot 605323-13536-00 Rev. 01Fluorochrome Molecule TypesSmaSome Common FluorochromesFluorochromeType of moleculeFluorescein isothyocyanate (FITC)Small organicAle

4、xa Fluor 488Small organicPhycoerythrin (PE)ProteinPE-CyTM5Protein tandemPeridinin chlorophyll protein (PerCP)ProteinPerCP-CyTM5.5Protein tandemPE-CyTM7Protein tandemAllophycocyanin (APC)ProteinAlexa Fluor 647Small organicAPC-CyTM7Protein tandemBDTM APC-H7Protein tandemBD HorizonTM V450Small organicP

5、acific BlueTMSmall organicBD HorizonTM V500Small organicAmCyanProtein423-13536-00 Rev. 01Some Common FluorochromesFluorSmall Organic FluorochromesAdvantagesLow molecular weightEasy to conjugatedirect attachment to free amino groups on mAbExcellent stabilityExtremely consistent emission spectraDisadv

6、antagesSmall Stokes Shift (50100 nm)Tend to be less bright523-13536-00 Rev. 01Small Organic FluorochromesAdvProtein FluorochromesAdvantagesGood stabilityConsistent emission spectraMedium Stokes Shift (75200 nm)Tend to be more brightDisadvantagesHigh molecular weightMore difficult to conjugateinterme

7、diaries needed to attach to mAb623-13536-00 Rev. 01Protein FluorochromesAdvantageTandem Dye FluorochromesAdvantagesVery large Stokes Shift (150300 nm)Tend to be very brightoften brighter than the fluorescent protein donor DisadvantagesHigh molecular weight (similar to fluorescent protein)Difficult t

8、o make consistently (lot-to-lot variation in emission properties)Harder to conjugate (same as fluorescent protein)Some tandems have poor stability723-13536-00 Rev. 01Tandem Dye FluorochromesAdvantNanocrystal FluorochromesAdvantagesLarge Stokes Shift (100500 nm)Tend to be very brightEmission peaks ar

9、e consistent and narrow, and do not change with variations in the excitation sourceHighly resistant to photobleachingNanocrystals share biophysical and conjugation properties DisadvantagesDifficult to conjugateInstability of bindingsCytotoxicityWide excitation range produces cross-laser spillover823

10、-13536-00 Rev. 01Nanocrystal FluorochromesAdvanExcitation and EmissionExcitation wavelengths determine lasers that can excite the fluorochrome.Emission wavelengths determine filters and PMTs that can measure the emission signal.923-13536-00 Rev. 01Excitation and EmissionExcitatKnow Your CytometerCyt

11、ometer ConfigurationBD FACSCantoTM II 4-2-2 configuration is shown below BDTM LSR II 4-2-2 configuration is similar4 detectors for blue laser 2 detectors for red laser 2 detectors for violet laser 1023-13536-00 Rev. 01Know Your CytometerCytometer CTypical Excitation and EmissionBD FACSCanto II 4-2-2

12、 (BD LSR II 4-2-2 is similar)Detector Range Violet Laser 405 nmBlue Laser 488 nmRed Laser 633 nm410490 nmBD Horizon V450 Pacific BlueTM500560 nmBD Horizon V500 AmCyan515545 nmFITCAlexa Fluor 488564606 nmPE650670 nmAPCAlexa Fluor 647670735 nmPerCP-Cy5.5PE-Cy5PerCP750810 nmPE-Cy7BD APC-H7APC-Cy71123-1

13、3536-00 Rev. 01Typical Excitation and EmissioFluorochrome Use Depends on the Cytometer Configuration 6-color 8-color More than 8 colorsFITC, Alexa Fluor 488FITC, Alexa Fluor 488FITC, Alexa Fluor 488PEPEPEPE-Texas Red, PE-Alexa Fluor 610, or PE-Alexa Fluor 594PE-Cy5, PerCP,or PerCP-Cy5.5PE-Cy5, PerCP

14、,or PerCP-Cy5.5PE-Cy5, PerCP,or PerCP-Cy5.5PE-Cy7PE-Cy7PE-Cy7APC, Alexa Fluor 647APC, Alexa Fluor 647APC, Alexa Fluor 647APC-Cy5.5, Alexa Fluor 680, Alexa Fluor 700APC-H7, APC-Cy7APC-H7, APC-Cy7APC-H7, APC-Cy7BDHorizon V500, AmCyanBDHorizon V500, AmCyanBDHorizon V450, Pacific BlueBD Horizon V450, Pa

15、cific BluePacific Orange, Q-dots1223-13536-00 Rev. 01Fluorochrome Use Depends on tFluorochrome BrightnessThe 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 photo

16、ns absorbed.Brightness = x QyRelative Brightness =Brightness of PEBrightness1323-13536-00 Rev. 01Fluorochrome BrightnessThe briSome Fluorochromes are MUCH BrighterPE is 50 x brighter than FITC and 10 x brighter than APC.APC is 5x brighter than Pacific Blue.Extinction coefficient is more significant

17、than quantum yield in determining brightness. FluorochromeMolar Extinction Coefficient (mol-1 x cm-1)Quantum YieldRelative Brightness (to PE)PE19600000.98100.00%PE-Cy519600000.881.63%PerCP320000NA16.66%APC2320000.688.21%FITC670000.50 1.74%Pacific Blue360000.801.5%1423-13536-00 Rev. 01Some Fluorochro

18、mes are MUCH BrDD = difference between the medians of the positive and negative populationsW = spread (2 x rSD) of the negative populationStain IndexStain Index =Stain index is a practical way to characterize the brightness of a marker with respect to a given optical configuration.DWWStain index can

19、 also be used to characterize the sensitivity of a fluoresence parameter.1523-13536-00 Rev. 01DD = difference between the meTypical CD4 Stain IndexesBD LSR II FluorochromeCD4 CloneFilterSetStain IndexRelative BrightnessPERPA-T4585/40305100.00%PE-Cy5RPA-T4695/4019881.63%PerCPRPA-T4695/403016.66%APCRP

20、A-T4660/202638.21%FITCRPA-T4530/30431.74%Pacific BlueRPA-T4440/40631.5%APC has a higher CD4 stain index than PE-Cy5, but approximately 1/10th the relative brightness.1623-13536-00 Rev. 01Typical CD4 Stain IndexesBD LTypical CD4 Stain IndexesBD FACSCanto II FluorochromeCD4 CloneFilterSetStain IndexRe

21、lative BrightnessPESK3585/42467100.00%PE-Cy581.63%PerCPSK3695/405916.66%APCSK3660/203848.21%FITCSK3530/30581.74%Pacific BlueSK3450/50131.5%FITC has approximately the same CD4 stain index as PerCP, but approximately 1/10th the relative brightness.1723-13536-00 Rev. 01Typical CD4 Stain IndexesBD FStai

22、n 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 index on your own system.1823-

23、13536-00 Rev. 01Stain Index Factors Stain indeCD4 Stain Indexes Across CytometersStain Index Exercise1923-13536-00 Rev. 01CD4 Stain Indexes Across CytomFluorescence SpilloverEmission of FITC in PE channel2023-13536-00 Rev. 01Fluorescence SpilloverEmissionSignificant Spillovers on 4-2-2 Configuration

24、2123-13536-00 Rev. 01Significant Spillovers on 4-2-Spillover Decreases SensitivityWithout CD45 AmCyanWith CD45 AmCyanCD19 FITCSpillover can significantly increase the variability of negative and dim populations, even after compensation is applied.2223-13536-00 Rev. 01Spillover Decreases SensitivitLo

25、st Population due to SpilloverLymphocytes stained with CD45 FITC and CD4 PECD45 FITC causes dim CD4+CD45+ to be difficult to distinguish due to significant FITC spillover into PE.Lymphocytes stained with CD45 PerCP and CD4 PECD45 PerCP allows dim CD4+CD45+to be distinguished from backgrounddue to mi

26、nimal PerCP spillover into PE.2323-13536-00 Rev. 01Lost Population due to SpillovTandem Dye IssuesUse 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 temperatur

27、e.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.2423-13536-00 Rev. 01Tandem Dye IssuesUse tandem dyTandem DegradationFalse PositivesFalse positives i

28、n APC channel reduced in absence of APC-Cy7With CD8 APC-Cy7WithoutCD8 APC-Cy72523-13536-00 Rev. 01Tandem DegradationFalse PositTandem Degradation Over Time0 hours2 hours20 hoursPECD3 PE-Cy5CD8 PE-Cy7Time Sample Left in LightPEPE2623-13536-00 Rev. 01Tandem Degradation Over Time0 APC-H7 Is More Stable

29、 Than APC-Cy7 Comparison of Sample Stability(in BD Stabilizing Fixative at RT)0501001502002500124682448Hours of light exposure% SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy72723-13536-00 Rev. 01APC-H7 Is More Stable Than APCTandem Dye RecommendationsWhen using APC-Cy7 and PE-Cy7, beware of fix

30、ative 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 APC-H7 conjugated antibodies that are more stable than APC-Cy7 conj

31、ugates and have less spillover into the APC detector.2823-13536-00 Rev. 01Tandem Dye RecommendationsWhenNew Violet Laser FluorochromesBD Horizon V450Maximum excitation at 404 nm, emission peak of 448 nm.BDHorizon V450 reagents exhibit performance comparable to Pacific Blue reagents as measured by St

32、ain Index.Improved stability with fixation compared to Pacific Blue.2923-13536-00 Rev. 01New Violet Laser FluorochromesNew Violet Laser FluorochromesBD Horizon V500Maximum excitation at 415 nm, emission peak of 500 nm.Provides significantly reduced spillover into the FITC channel compared to AmCyanV

33、500 has low excitation with the blue laser.Improved stability with fixation compared to AmCyan.3023-13536-00 Rev. 01New Violet Laser FluorochromesBD Horizon V500 Low Spillover into FITCV500 stained cellsAmCyan stained cellsData is uncompensated.3123-13536-00 Rev. 01BD Horizon V500 Low Spillover Fact

34、ors Affecting Reagent PerformanceRelative brightness of the fluorochromeNumber of fluorochromes per antibodyExpression levels on (or in) the cells of interestBackground contributionsSpilloverPhotostabilityReagent environmentCytometer configuration3223-13536-00 Rev. 01Factors Affecting Reagent PerfNon-Conjugated Fluorescent DyesThese dyes bind directly to cell components.Viability dyes discrim