流式细胞技术荧光抗体.ppt

Fluorochromes 23-13536-00 Rev.01 1 Fluorochrome Properties Desirable 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 wavelengths Relative brightness Fluorochromes 23-13536-00 Rev.01 2 Fluorochrome Molecule Types Small organic molecules examples:FITC,BD HorizonTM V450,Cy7 Fluorescent proteins examples:PE,APC,PerCP Tandem dyestypically,the coupling of a fluorescent protein donor with a small organic molecule acceptor examples:PE-Cy7,PerCP-CyTM5.5 Nanocrystals(Qdots)inorganic semiconductors examples:Qdot 565,Qdot 605 Fluorochromes 23-13536-00 Rev.01 3 Some Common Fluorochromes Fluorochrome Type of molecule Fluorescein isothyocyanate(FITC)Small organic Alexa Fluor 488 Small organic Phycoerythrin(PE)Protein PE-CyTM5 Protein tandem Peridinin chlorophyll protein(PerCP)Protein PerCP-CyTM5.5 Protein tandem PE-CyTM7 Protein tandem Allophycocyanin(APC)Protein Alexa Fluor 647 Small organic APC-CyTM7 Protein tandem BDTM APC-H7 Protein tandem BD HorizonTM V450 Small organic Pacific BlueTM Small organic BD HorizonTM V500 Small organic AmCyan Protein Fluorochromes 23-13536-00 Rev.01 4 Small Organic Fluorochromes Advantages Low molecular weight Easy to conjugatedirect attachment to free amino groups on mAb Excellent stability Extremely consistent emission spectra Disadvantages Small Stokes Shift(50100 nm)Tend to be less bright Fluorochromes 23-13536-00 Rev.01 5 Protein Fluorochromes Advantages Good stability Consistent emission spectra Medium Stokes Shift(75200 nm)Tend to be more bright Disadvantages High molecular weight More difficult to conjugateintermediaries needed to attach to mAb Fluorochromes 23-13536-00 Rev.01 6 Tandem Dye Fluorochromes Advantages Very large Stokes Shift(150300 nm)Tend to be very brightoften brighter than 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 stability Fluorochromes 23-13536-00 Rev.01 7 Nanocrystal Fluorochromes Advantages 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 Instability of bindings Cytotoxicity Wide excitation range produces cross-laser spillover Fluorochromes 23-13536-00 Rev.01 8 Excitation and Emission Excitation wavelengths determine lasers that can excite the fluorochrome.Emission wavelengths determine filters and PMTs that can measure the emission signal.Fluorochromes 23-13536-00 Rev.01 9 Know Your Cytometer Cytometer Configuration BD FACSCantoTM II 4-2-2 configuration is shown below BDTM LSR II 4-2-2 configuration is similar 4 detectors for blue laser 2 detectors for red laser 2 detectors for violet laser Fluorochromes 23-13536-00 Rev.01 10 Typical Excitation and Emission BD FACSCanto II 4-2-2(BD LSR II 4-2-2 is similar)Detector Range Violet Laser 405 nm Blue Laser 488 nm Red Laser 633 nm 410490 nm BD Horizon V450 Pacific BlueTM 500560 nm BD Horizon V500 AmCyan 515545 nm FITC Alexa Fluor 488 564606 nm PE 650670 nm APC Alexa Fluor 647 670735 nm PerCP-Cy5.5 PE-Cy5 PerCP 750810 nm PE-Cy7 BD APC-H7 APC-Cy7 Fluorochromes 23-13536-00 Rev.01 11 Fluorochrome Use Depends on the Cytometer Configuration 6-color 8-color More than 8 colors FITC,Alexa Fluor 488 FITC,Alexa Fluor 488 FITC,Alexa Fluor 488 PE PE PE PE-Texas Red,PE-Alexa Fluor 610,or PE-Alexa Fluor 594 PE-Cy5,PerCP,or PerCP-Cy5.5 PE-Cy5,PerCP,or PerCP-Cy5.5 PE-Cy5,PerCP,or PerCP-Cy5.5 PE-Cy7 PE-Cy7 PE-Cy7 APC,Alexa Fluor 647 APC,Alexa Fluor 647 APC,Alexa Fluor 647 APC-Cy5.5,Alexa Fluor 680,Alexa Fluor 700 APC-H7,APC-Cy7 APC-H7,APC-Cy7 APC-H7,APC-Cy7 BDHorizon V500,AmCyan BDHorizon V500,AmCyan BDHorizon V450,Pacific Blue BD Horizon V450,Pacific Blue Pacific Orange,Q-dots Fluorochromes 23-13536-00 Rev.01 12 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 emitted to photons absorbed.Brightness=x Qy Relative Brightness=Brightness of PE Brightness Fluorochromes 23-13536-00 Rev.01 13 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 is more significant than quantum yield in determining brightness.Fluorochrome Molar Extinction Coefficient(mol-1 x cm-1)Quantum Yield Relative Brightness(to PE)PE 1960000 0.98 100.00%PE-Cy5 1960000 0.8 81.63%PerCP 320000 NA 16.66%APC 232000 0.68 8.21%FITC 67000 0.50 1.74%Pacific Blue 36000 0.80 1.5%Fluorochromes 23-13536-00 Rev.01 14 D D=differen