1、Fluorochromes23-13536-00 Rev.01Fluorochrome PropertiesDesirable properties for fluorochromes:High relative brightness Narrow emission spectrum(low spectral overlap in combination)Easily conjugated(for immunophenotyping)Fluorochromes can be characterized by:Type of molecule Excitation and emission wa
2、velengths Relative brightness223-13536-00 Rev.01Fluorochrome Molecule Types Small organic moleculesexamples:FITC,BD HorizonTM V450,Cy7 Fluorescent proteinsexamples:PE,APC,PerCP Tandem dyestypically,the coupling of a fluorescent protein donor with a small organic molecule acceptorexamples:PE-Cy7,PerC
3、P-CyTM5.5 Nanocrystals(Qdots)inorganic semiconductorsexamples:Qdot 565,Qdot 605323-13536-00 Rev.01Some Common FluorochromesFluorochromeType of moleculeFluorescein isothyocyanate(FITC)Small organicAlexa Fluor 488Small organicPhycoerythrin(PE)ProteinPE-CyTM5Protein tandemPeridinin chlorophyll protein(
4、PerCP)ProteinPerCP-CyTM5.5Protein tandemPE-CyTM7Protein tandemAllophycocyanin(APC)ProteinAlexa Fluor 647Small organicAPC-CyTM7Protein tandemBDTM APC-H7Protein tandemBD HorizonTM V450Small organicPacific BlueTMSmall organicBD HorizonTM V500Small organicAmCyanProtein423-13536-00 Rev.01Small Organic Fl
5、uorochromesAdvantages Low molecular weight Easy to conjugatedirect attachment to free amino groups on mAb Excellent stability Extremely consistent emission spectraDisadvantages Small Stokes Shift(50100 nm)Tend to be less bright523-13536-00 Rev.01Protein FluorochromesAdvantages Good stability Consist
6、ent emission spectra Medium Stokes Shift(75200 nm)Tend to be more brightDisadvantages High molecular weight More difficult to conjugateintermediaries needed to attach to mAb623-13536-00 Rev.01Tandem Dye FluorochromesAdvantages Very large Stokes Shift(150300 nm)Tend to be very brightoften brighter th
7、an the fluorescent protein donor Disadvantages High 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 tandems have poor stability723-13536-00 Rev.01Nanocrystal FluorochromesA
8、dvantages Large Stokes Shift(100500 nm)Tend to be very bright Emission peaks are consistent and narrow,and do not change with variations in the excitation source Highly resistant to photobleaching Nanocrystals share biophysical and conjugation properties Disadvantages Difficult to conjugate Instabil
9、ity of bindings Cytotoxicity Wide excitation range produces cross-laser spillover823-13536-00 Rev.01Excitation and Emission Excitation wavelengths determine lasers that can excite the fluorochrome.Emission wavelengths determine filters and PMTs that can measure the emission signal.923-13536-00 Rev.0
10、1Know Your CytometerCytometer 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.01Typical Excitation and EmissionBD FACSCanto II 4-2-2(BD LSR
11、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-Cy71123-13536-00 R
12、ev.01Fluorochrome 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,or PerCP-Cy5.5PE-Cy5,PerCP,or PerCP-Cy5.5PE-Cy
13、7PE-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,Pacific BluePacific Orange,Q-dots1223-13536-00 Rev.01Fluorochr
14、ome 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 photons absorbed.Brightness=x QyRelative Brightness=Brightness of PEBrightness1323-13536-00 Rev.01So
15、me 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.FluorochromeMolar Extinction Coefficient(mol-1 x cm-1)Quantum YieldRelative Brightn
16、ess(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.01DD=difference between the medians of the positive and negative populationsW=spread(2 x rSD)of the negative populationStain IndexStain Index=Stain i
17、ndex is a practical way to characterize the brightness of a marker with respect to a given optical configuration.DWWStain index can also be used to characterize the sensitivity of a fluoresence parameter.1523-13536-00 Rev.01Typical CD4 Stain IndexesBD LSR II FluorochromeCD4 CloneFilterSetStain Index
18、Relative BrightnessPERPA-T4585/40305100.00%PE-Cy5RPA-T4695/4019881.63%PerCPRPA-T4695/403016.66%APCRPA-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 Sta
19、in IndexesBD FACSCanto II FluorochromeCD4 CloneFilterSetStain IndexRelative 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 relati
20、ve brightness.1723-13536-00 Rev.01Stain 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 signal Dont depend on published valuesmeasu
21、re stain index on your own system.1823-13536-00 Rev.01CD4 Stain Indexes Across CytometersStain Index Exercise1923-13536-00 Rev.01Fluorescence SpilloverEmission of FITC in PE channel2023-13536-00 Rev.01Significant Spillovers on 4-2-2 Configuration2123-13536-00 Rev.01Spillover Decreases SensitivityWit
22、hout CD45 AmCyanWith CD45 AmCyanCD19 FITCSpillover can significantly increase the variability of negative and dim populations,even after compensation is applied.2223-13536-00 Rev.01Lost Population due to SpilloverLymphocytes stained with CD45 FITC and CD4 PECD45 FITC causes dim CD4+CD45+to be diffic
23、ult 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 minimal PerCP spillover into PE.2323-13536-00 Rev.01Tandem Dye IssuesUse tandem dyes in research applications with consi
24、deration 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 of the parent dye(APC or PE).APC-H7,APC-Cy7,and PE-Cy7 perfor
25、mance can be affected by polystyrene tubes.2423-13536-00 Rev.01Tandem DegradationFalse PositivesFalse positives in APC channel reduced in absence of APC-Cy7With CD8 APC-Cy7WithoutCD8 APC-Cy72523-13536-00 Rev.01Tandem Degradation Over Time0 hours2 hours20 hoursPECD3 PE-Cy5CD8 PE-Cy7Time Sample Left i
26、n LightPEPE2623-13536-00 Rev.01APC-H7 Is More Stable 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.01Tandem Dye Recommendations When using APC-Cy7 and
27、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 APC-H7 conjugated antibodies that are more stable t
28、han APC-Cy7 conjugates and have less spillover into the APC detector.2823-13536-00 Rev.01New Violet Laser FluorochromesBD Horizon V450 Maximum excitation at 404 nm,emission peak of 448 nm.BD Horizon V450 reagents exhibit performance comparable to Pacific Blue reagents as measured by Stain Index.Impr
29、oved stability with fixation compared to Pacific Blue.2923-13536-00 Rev.01New Violet Laser FluorochromesBD Horizon V500 Maximum excitation at 415 nm,emission peak of 500 nm.Provides significantly reduced spillover into the FITC channel compared to AmCyanV500 has low excitation with the blue laser.Im
30、proved stability with fixation compared to AmCyan.3023-13536-00 Rev.01BD Horizon V500 Low Spillover into FITCV500 stained cellsAmCyan stained cellsData is uncompensated.3123-13536-00 Rev.01Factors Affecting Reagent Performance Relative brightness of the fluorochrome Number of fluorochromes per antib
31、ody Expression levels on(or in)the cells of interest Background contributions Spillover Photostability Reagent environment Cytometer configuration3223-13536-00 Rev.01Non-Conjugated Fluorescent Dyes These dyes bind directly to cell components.Viability dyes discriminate live cells from dead cells.exa
32、mples: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.Reporter dyes are used to study gene expression.examples:mCherry and G
33、FP.3323-13536-00 Rev.01Non-Conjugated Fluorescence Dye DetectionBD FACSCanto II 4-2-2 and BD LSR II 4-2-2Detector Range Violet Laser 405 nmBlue Laser 488 nmRed Laser 635 nm410490 nmHoechst 33342,DAPI,Horizon VPD 450,LIVE/DEAD Violet500560 nmLIVE/DEAD Aqua515545 nmCSFE,GFP,LIVE/DEAD Green564606 nmPI6
34、50670 nmTO-PRO-3670735 nm7-AAD750810 nmLIVE/DEAD Near-IRmCherry is best detected with a 561-nm laser option and a 590630 detector range.3423-13536-00 Rev.01BD Fluorescence Spectrum Viewerbdbiosciences/spectra Use the Fluorescence Spectrum Viewer to help choose fluorochromes based on:the number of colors you need in your experiment your cytometer configuration spillover issues3523-13536-00 Rev.01
