流式细胞技术荧光抗体

1、流式细胞技术荧光抗体23-13536-00 Rev. 01第1页，共35页，2022年，5月20日，11点21分，星期四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 molec

2、uleExcitation and emission wavelengthsRelative brightness223-13536-00 Rev. 01第2页，共35页，2022年，5月20日，11点21分，星期四Fluorochrome 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. 01第3页，共35页，2022年，5月20日，11点21分，星期四Some 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. 01第4页，共35页，2022年，5月20日，11点21分，星期四Small Organic FluorochromesAdvantagesLow molecular weightEasy to conjugatedirect attachment to free amino groups on mAbExcellent stabilityExtremely consistent emission spectraDisad

6、vantagesSmall Stokes Shift (50100 nm)Tend to be less bright523-13536-00 Rev. 01第5页，共35页，2022年，5月20日，11点21分，星期四Protein FluorochromesAdvantagesGood stabilityConsistent emission spectraMedium Stokes Shift (75200 nm)Tend to be more brightDisadvantagesHigh molecular weightMore difficult to conjugateinter

7、mediaries needed to attach to mAb623-13536-00 Rev. 01第6页，共35页，2022年，5月20日，11点21分，星期四Tandem 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)Difficul

8、t 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. 01第7页，共35页，2022年，5月20日，11点21分，星期四Nanocrystal FluorochromesAdvantagesLarge Stokes Shift (100500 nm)Tend to be very brightEmission peak

9、s are 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 spillove

10、r823-13536-00 Rev. 01第8页，共35页，2022年，5月20日，11点21分，星期四Excitation 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. 01第9页，共35页，2022年，5月20日，11点21分，星期四Know Your Cytome

11、terCytometer 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. 01第10页，共35页，2022年，5月20日，11点21分，星期四Typical Excitation and EmissionBD FACSCanto

12、II 4-2-2 (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-C

13、y71123-13536-00 Rev. 01第11页，共35页，2022年，5月20日，11点21分，星期四Fluorochrome 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-

14、Cy5, PerCP,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 Horizo

15、n V450, Pacific BluePacific Orange, Q-dots1223-13536-00 Rev. 01第12页，共35页，2022年，5月20日，11点21分，星期四Fluorochrome 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 emi

16、tted to photons absorbed.Brightness = x QyRelative Brightness =Brightness of PEBrightness1323-13536-00 Rev. 01第13页，共35页，2022年，5月20日，11点21分，星期四Some 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 mo

17、re significant 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. 01

18、第14页，共35页，2022年，5月20日，11点21分，星期四DD = 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.D

19、WWStain index can also be used to characterize the sensitivity of a fluoresence parameter.1523-13536-00 Rev. 01第15页，共35页，2022年，5月20日，11点21分，星期四Typical CD4 Stain IndexesBD LSR II FluorochromeCD4 CloneFilterSetStain IndexRelative BrightnessPERPA-T4585/40305100.00%PE-Cy5RPA-T4695/4019881.63%PerCPRPA-T4

20、695/403016.66%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. 01第16页，共35页，2022年，5月20日，11点21分，星期四Typical CD4 Stain IndexesBD FACSCanto II FluorochromeCD4 Clone

21、FilterSetStain 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 relative brightness.1723-13536-00 Rev. 01第17页，共35页，2

22、022年，5月20日，11点21分，星期四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 index

23、 on your own system.1823-13536-00 Rev. 01第18页，共35页，2022年，5月20日，11点21分，星期四CD4 Stain Indexes Across CytometersStain Index Exercise1923-13536-00 Rev. 01第19页，共35页，2022年，5月20日，11点21分，星期四Fluorescence SpilloverEmission of FITC in PE channel2023-13536-00 Rev. 01第20页，共35页，2022年，5月20日，11点21分，星期四Significant Sp

24、illovers on 4-2-2 Configuration2123-13536-00 Rev. 01第21页，共35页，2022年，5月20日，11点21分，星期四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. 0

25、1第22页，共35页，2022年，5月20日，11点21分，星期四Lost 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 dis

26、tinguished from backgrounddue to minimal PerCP spillover into PE.2323-13536-00 Rev. 01第23页，共35页，2022年，5月20日，11点21分，星期四Tandem 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 lig

27、ht, 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.2423-13536-00 Rev. 01第24页，共35页，2022年，5月20日，11点21分，星期四Tandem Degr

28、adationFalse PositivesFalse positives in APC channel reduced in absence of APC-Cy7With CD8 APC-Cy7WithoutCD8 APC-Cy72523-13536-00 Rev. 01第25页，共35页，2022年，5月20日，11点21分，星期四Tandem Degradation Over Time0 hours2 hours20 hoursPECD3 PE-Cy5CD8 PE-Cy7Time Sample Left in LightPEPE2623-13536-00 Rev. 01第26页，共35页

29、，2022年，5月20日，11点21分，星期四APC-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. 01第27页，共35页，2022年，5月20日，11点21分，星期四Tandem Dye Recommendatio

30、nsWhen 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 APC-H7 conjugated ant

31、ibodies that are more stable than APC-Cy7 conjugates and have less spillover into the APC detector.2823-13536-00 Rev. 01第28页，共35页，2022年，5月20日，11点21分，星期四New Violet Laser FluorochromesBD Horizon V450Maximum excitation at 404 nm, emission peak of 448 nm.BDHorizon V450 reagents exhibit performance compa

32、rable to Pacific Blue reagents as measured by Stain Index.Improved stability with fixation compared to Pacific Blue.2923-13536-00 Rev. 01第29页，共35页，2022年，5月20日，11点21分，星期四New Violet Laser FluorochromesBD Horizon V500Maximum excitation at 415 nm, emission peak of 500 nm.Provides significantly reduced s

33、pillover into the FITC channel compared to AmCyanV500 has low excitation with the blue laser.Improved stability with fixation compared to AmCyan.3023-13536-00 Rev. 01第30页，共35页，2022年，5月20日，11点21分，星期四BD Horizon V500 Low Spillover into FITCV500 stained cellsAmCyan stained cellsData is uncompensated.312

34、3-13536-00 Rev. 01第31页，共35页，2022年，5月20日，11点21分，星期四Factors 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. 01第32页，共35页，2022年，5月20日，11点21分，星期四Non-Conjugated Fluorescent DyesThese dyes bind directly to cell components.Viability