流式細(xì)胞技術(shù)熒光抗體課件

1、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 moleculeExcitation and emission wavelengthsRelative brightness第一頁(yè)

2、，共三十五頁(yè)。1流式細(xì)胞技術(shù)熒光抗體Fluorochrome Molecule TypesSmall organic moleculesexamples: FITC, BD HorizonTM V450, Cy7Fluorescent proteinsexamples: PE, APC, PerCPTandem dyestypically, the coupling of a fluorescent protein donor with a small organic molecule acceptorexamples: PE-Cy7, PerCP-CyTM5.5Nanocrystals (Q

3、dots)inorganic semiconductorsexamples: Qdot 565, Qdot 605第二頁(yè)，共三十五頁(yè)。2流式細(xì)胞技術(shù)熒光抗體Some Common FluorochromesFluorochromeType of moleculeFluorescein isothyocyanate (FITC)Small organicAlexa Fluor 488Small organicPhycoerythrin (PE)ProteinPE-CyTM5Protein tandemPeridinin chlorophyll protein (PerCP)ProteinPerC

4、P-CyTM5.5Protein tandemPE-CyTM7Protein tandemAllophycocyanin (APC)ProteinAlexa Fluor 647Small organicAPC-CyTM7Protein tandemBDTM APC-H7Protein tandemBD HorizonTM V450Small organicPacific BlueTMSmall organicBD HorizonTM V500Small organicAmCyanProtein第三頁(yè)，共三十五頁(yè)。3流式細(xì)胞技術(shù)熒光抗體Small Organic FluorochromesAdv

5、antagesLow molecular weightEasy to conjugatedirect attachment to free amino groups on mAbExcellent stabilityExtremely consistent emission spectraDisadvantagesSmall Stokes Shift (50100 nm)Tend to be less bright第四頁(yè)，共三十五頁(yè)。4流式細(xì)胞技術(shù)熒光抗體Protein FluorochromesAdvantagesGood stabilityConsistent emission spect

6、raMedium Stokes Shift (75200 nm)Tend to be more brightDisadvantagesHigh molecular weightMore difficult to conjugateintermediaries needed to attach to mAb第五頁(yè)，共三十五頁(yè)。5流式細(xì)胞技術(shù)熒光抗體Tandem Dye FluorochromesAdvantagesVery large Stokes Shift (150300 nm)Tend to be very brightoften brighter than the fluorescent

7、 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 tandems have poor stability第六頁(yè)，共三十五頁(yè)。6流式細(xì)胞技術(shù)熒光抗體Nanocrystal FluorochromesAdvantagesLarge

8、 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 photobleachingNanocrystals share biophysical and conjugation properties DisadvantagesDifficult to conjugateInstability of bindingsCyt

9、otoxicityWide excitation range produces cross-laser spillover第七頁(yè)，共三十五頁(yè)。7流式細(xì)胞技術(shù)熒光抗體Excitation and EmissionExcitation wavelengths determine lasers that can excite the fluorochrome.Emission wavelengths determine filters and PMTs that can measure the emission signal.第八頁(yè)，共三十五頁(yè)。8流式細(xì)胞技術(shù)熒光抗體Know Your Cytome

10、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 第九頁(yè)，共三十五頁(yè)。9流式細(xì)胞技術(shù)熒光抗體Typical Excitation and EmissionBD FACSCanto II 4-2-2 (BD LSR II 4-2-2 is sim

11、ilar)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-Cy7第十頁(yè)，共三十五頁(yè)。10流式細(xì)胞技術(shù)熒光抗體Fluoroch

12、rome 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-Cy7PE-C

13、y7PE-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-dots第十一頁(yè)，共三十五頁(yè)。11流式細(xì)胞技術(shù)熒光

14、抗體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 emitted to photons absorbed.Brightness = x QyRelative Brightness =Brightness of PEBrightness第十二頁(yè)

15、，共三十五頁(yè)。12流式細(xì)胞技術(shù)熒光抗體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 more significant than quantum yield in determining brightness. FluorochromeMolar Extinction Coefficient (mol-1 x cm-1)Quantum

16、 YieldRelative Brightness (to PE)PE19600000.98100.00%PE-Cy519600000.881.63%PerCP320000NA16.66%APC2320000.688.21%FITC670000.50 1.74%Pacific Blue360000.801.5%第十三頁(yè)，共三十五頁(yè)。13流式細(xì)胞技術(shù)熒光抗體DD = difference between the medians of the positive and negative populationsW = spread (2 x rSD) of the negative populati

17、onStain 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 also be used to characterize the sensitivity of a fluoresence parameter.第十四頁(yè)，共三十五頁(yè)。14流式細(xì)胞技術(shù)熒光抗體Typical CD4 Stain IndexesBD LSR II Fluor

18、ochromeCD4 CloneFilterSetStain IndexRelative 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 brightnes

19、s.第十五頁(yè)，共三十五頁(yè)。15流式細(xì)胞技術(shù)熒光抗體Typical CD4 Stain 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 P

20、erCP, but approximately 1/10th the relative brightness.第十六頁(yè)，共三十五頁(yè)。16流式細(xì)胞技術(shù)熒光抗體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

21、 signalDont depend on published valuesmeasure stain index on your own system.第十七頁(yè)，共三十五頁(yè)。17流式細(xì)胞技術(shù)熒光抗體CD4 Stain Indexes Across CytometersStain Index Exercise第十八頁(yè)，共三十五頁(yè)。18流式細(xì)胞技術(shù)熒光抗體Fluorescence SpilloverEmission of FITC in PE channel第十九頁(yè)，共三十五頁(yè)。19流式細(xì)胞技術(shù)熒光抗體Significant Spillovers on 4-2-2 Configuration第二

22、十頁(yè)，共三十五頁(yè)。20流式細(xì)胞技術(shù)熒光抗體Spillover Decreases SensitivityWithout CD45 AmCyanWith CD45 AmCyanCD19 FITCSpillover can significantly increase the variability of negative and dim populations, even after compensation is applied.第二十一頁(yè)，共三十五頁(yè)。21流式細(xì)胞技術(shù)熒光抗體Lost Population due to SpilloverLymphocytes stained with CD

23、45 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 minimal PerCP spillover into PE.第二十二頁(yè)，共三十五頁(yè)。22流式細(xì)胞技術(shù)熒光抗體Tan

24、dem 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 temperature.Degradation causes lower emission in the Cy7 detector and higher emission in the d

25、etector of the parent dye (APC or PE).APC-H7, APC-Cy7, and PE-Cy7 performance can be affected by polystyrene tubes.第二十三頁(yè)，共三十五頁(yè)。23流式細(xì)胞技術(shù)熒光抗體Tandem DegradationFalse PositivesFalse positives in APC channel reduced in absence of APC-Cy7With CD8 APC-Cy7WithoutCD8 APC-Cy7第二十四頁(yè)，共三十五頁(yè)。24流式細(xì)胞技術(shù)熒光抗體Tandem Deg

26、radation Over Time0 hours2 hours20 hoursPECD3 PE-Cy5CD8 PE-Cy7Time Sample Left in LightPEPE第二十五頁(yè)，共三十五頁(yè)。25流式細(xì)胞技術(shù)熒光抗體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

27、-Cy7CD8 APC-Cy7第二十六頁(yè)，共三十五頁(yè)。26流式細(xì)胞技術(shù)熒光抗體Tandem Dye RecommendationsWhen 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 inst

28、ability than APC-Cy7 and PE-Cy7.BD offers APC-H7 conjugated antibodies that are more stable than APC-Cy7 conjugates and have less spillover into the APC detector.第二十七頁(yè)，共三十五頁(yè)。27流式細(xì)胞技術(shù)熒光抗體New Violet Laser FluorochromesBD Horizon V450Maximum excitation at 404 nm, emission peak of 448 nm.BDHorizon V450

29、reagents exhibit performance comparable to Pacific Blue reagents as measured by Stain Index.Improved stability with fixation compared to Pacific Blue.第二十八頁(yè)，共三十五頁(yè)。28流式細(xì)胞技術(shù)熒光抗體New Violet Laser FluorochromesBD Horizon V500Maximum excitation at 415 nm, emission peak of 500 nm.Provides significantly redu

30、ced spillover into the FITC channel compared to AmCyanV500 has low excitation with the blue laser.Improved stability with fixation compared to AmCyan.第二十九頁(yè)，共三十五頁(yè)。29流式細(xì)胞技術(shù)熒光抗體BD Horizon V500 Low Spillover into FITCV500 stained cellsAmCyan stained cellsData is uncompensated.第三十頁(yè)，共三十五頁(yè)。30流式細(xì)胞技術(shù)熒光抗體Fact

31、ors Affecting Reagent PerformanceRelative brightness of the fluorochromeNumber of fluorochromes per antibodyExpression levels on (or in) the cells of interestBackground contributionsSpilloverPhotostabilityReagent environmentCytometer configuration第三十一頁(yè)，共三十五頁(yè)。31流式細(xì)胞技術(shù)熒光抗體Non-Conjugated Fluorescent DyesThese 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 dyes are used to study cell division. examples: CSFE, PKH 67, and BD Horizon VPD 450.DNA dyes are used for