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Flow CytometryFluorophore selection guidefor flow cytometryDye choices for your instrument made easy

Fluorophore selection guide for flow cytometryDyes for the 405 nm and 407 nm lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Dyes for the 488 nm laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Dyes for the 635 nm laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Accuri C6 Flow Cytometer System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6BD FACSCalibur System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7BD FACScan Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8BD FACSCanto System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9BD FACSCanto II System, 2-laser option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11BD FACSCanto II System, 3-laser option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13BD LSR Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15BD LSR II Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17BD FACSAria Cell Sorter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19BD FACSArray Bioanalyzer System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21BD FACSVantage SE Cell Sorter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Beckman Coulter Cytomics FC 500 Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Beckman Coulter EPICS XL Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Beckman Coulter Cell Lab Quanta SC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Beckman Coulter EPICS ALTRA Cell Sorter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Beckman Coulter CyAn ADP Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Beckman Coulter MoFlo XDP Cell Sorter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Guava Personal Cell Analysis (PCA) System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Guava EasyCyte Mini System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Guava EasyCyte Plus System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41www.invitrogen.com

Flow CytometryFluorophore selection guideDyes for the 405 nm and 407 nm lasersPacific Blue The dye of choice for the first violet channel Excited optimally at 410 nm with emission maximum at 455 nm No UV excitation Minimal spectral overlap with green fluorophoresAlexa Fluor 405 Excited optimally at 402 nm with emission maximum at 421 nm Minimal spectral overlap with green fluorophores Lower in fluorescence than Pacific Blue dye, higher background in some intracellular applications May withstand fixation better than Pacific Blue dye under some circumstances Some UV excitationPacific Orange Compatible with Pacific Blue dye for multicolor applications of the violet laser Excited optimally at 405 nm with emission maximum at 551 nm Best signal obtained with an RPE emission filter (i.e., 548/42 or 575/26)Qdot 525nanocrystalQdot 565nanocrystalQdot 605nanocrystalQdot 655nanocrystal Allows high level multiplexing with a single excitation source Ultimate in photostability Excited optimally with UV or violet Also excited by every laser below the emission wavelength Emission centered on the wavelength in nanocrystal name May require cross-laser compensation depending on fluorophores used Can replace selected energy transfer (tandem) conjugates2Qdot 705nanocrystalQdot 800nanocrystal

Fluorophore selection guideDyes for the 488 nm laserAlexa Fluor 488 Brighter and more photostable than FITC Interchangeable with FITC with only minor changes in compensation Excited at 488 nm with emission maximum at 519 nm pH insensitive Ideal primary color for flow cytometryRPE (R-Phycoerythrin) Excited at 488 nm or 532 nm with emission maximum at 575 nm Very bright fluorescence Shows some excitation with 405 nm laser Highly susceptible to photobleachingRPE-Texas Red Energy transfer dye excited at 488 nm with emission maximum at 615 nm Can be measured in FL3 channel on BD and Beckman Coulter instrumentsRPE-Alexa Fluor 610 Energy transfer dye excited at 488 nm with emission maximum at 628 nm Alternative to RPE-Texas Red dye Higher quantum yield than RPE-Texas Red dye with lower compensation against RPE Ideal for use with RPE-Alexa Fluor 700 or RPE-Cy 5.5 dyes in multicolor panels Can be measured in FL3 channel on BD and Beckman Coulter instruments Requires filter modification for optimal use on Beckman Coulter EPICS XL instrumentTRI-COLOR Energy transfer dye excited at 488 nm with emission maximum at 670 nm Also known as PE-Cy 5 Bright fluorescence Low compensation against RPE Significant emission in APC channel with 633 nm excitation Measured in FL3 channel on BD FACScan and BD FACSCalibur instruments Measured in FL4 channel on Beckman Coulter EPICS XL instrument Can demonstrate monocyte binding in some circumstancescontinued3www.invitrogen.com

Flow CytometryFluorophore selection guideDyes for the 488 nm laser, continuedRPE-Alexa Fluor 700 Fluorochrome of choice for BD FACSCalibur instrument Energy transfer dye excited at 488 nm with emission maximum at 723 nm Very low compensation against FL2 and FL4 when detected in FL3 on BD FACSCalibur or BD FACScan instrument Bright alternative to RPE-Cy 5 (TRI-COLOR , CyChrome, PC5), PerCP, and RPE-Cy 5.5 Together with RPE-Alexa Fluor 610 dye, constitutes the optimal four-color combination using the 488 nm laser lineRPE-Cy 5.5 Energy transfer dye excited at 488 nm with emission maximum at 694 nm Lower photostability than RPE-Alexa Fluor 700 Requires more compensation against APC channel than RPE-Alexa Fluor 700 Measured in FL3 channel on BD FACScan and BD FACSCalibur instruments Measured in FL4 on Beckman Coulter EPICS XL instrumentRPE-Cy 7 Provides long-wavelength emission off the 488 nm laser Energy transfer dye excited at 488 nm with emission maximum at 775 nm Requires varying degrees of compensation out of RPE channel4

Fluorophore selection guideDyes for the 635 nm laserAlexa Fluor 647 Energy transfer dye excited at 633/635 nm with emission maximum at 668 nm An excellent alternative to APC and Cy 5; potentially less background with intracellular staining Brightness comparable to APC in flow cytometry Significantly more photostable than APC and Cy 5Alexa Fluor 700 Energy transfer dye excited at 633/635 nm with emission maximum at 723 nm Allows three-color analysis using a red laser when combined with APC and APC-Alexa Fluor 750 Extremely bright and photostable A single, small-molecule direct conjugate alternative to the tandem APC-Cy 5.5 Minimal compensation against APC Requires significant compensation against RPE-Alexa Fluor 700 and RPE-Cy 5.5APC-Alexa Fluor 750 Energy transfer dye excited at 633/635 nm with emission maximum at 775 nm Significantly more photostable than APC-Cy 7 Lower compensation when paired with APC than is needed with the APC-Cy 7/APC pair Brighter and more stabile than APC-Cy 7 Perfectly interchangeable with APC-Cy 7 due to spectral similarity between Alexa Fluor 750 dye and Cy 75www.invitrogen.com

Flow CytometryFluorophore selection guideInstrument: Accuri C6 Flow Cytometer nelDefault filter Filter range (nm)FL1 Green530/30515–545FluorophoresOther fluorescent dyesAlexa Fluor 488Click-iT EdU Alexa Fluor 488FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1CFSEGFPJC-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1Argon488FL2 Yellow585/40565–605RPEPIAlexa Fluor 546Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalJC-1 or DiOC2(3)SNARF (low pH)pHrodo dyeArgon488FL3 Red675LP 675RPE-Alexa Fluor 700PIQdot 705 nanocrystal7-AADQdot 800 nanocrystalSNARF (high pH)RPE-Cy 5.5JC-1 or DiOC2(3)TRI-COLOR LIVE/DEAD Fixable Red Dead Cell StainRPE-Cy 7RPE-Alexa Fluor 750PerCPHe–Ne640FL4 Red675/24663–687APCClick-iT EdU Alexa Fluor 647Alexa Fluor 647LIVE/DEAD Fixable Far Red Dead Cell StainQdot 655 nanocrystalTO-PRO -3SYTOX RedMitoProbe DiIC1(5)CellTrace Far Red DDAO-SEBold recommended fluorophore.6

Fluorophore selection guideInstrument: BD FACSCalibur 488FL1 GreenDefault filter Filter range (nm)530/30515–545FluorophoresOther fluorescent dyesAlexa Fluor 488Click-iT EdU Alexa Fluor 488FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1CFSEGFPJC-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1Argon488FL2 Yellow585/42564–606RPEPIAlexa Fluor 546Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalJC-1 or DiOC2(3)SNARF (low pH)pHrodo dyeArgon488FL3 Red670LP 670RPE-Alexa Fluor 700PIQdot 705 nanocrystal7-AADQdot 800 nanocrystalSNARF (high pH)RPE-Cy 5.5JC-1 or DiOC2(3)TRI-COLOR LIVE/DEAD Fixable Red Dead Cell StainRPE-Cy 7RPE-Alexa Fluor 750PerCPHe–Ne635FL4 Red661/12656–667APCClick-iT EdU Alexa Fluor 647Alexa Fluor 647LIVE/DEAD Fixable Far Red Dead Cell StainQdot 655 nanocrystalTO-PRO -3SYTOX RedMitoProbe DiIC1(5)CellTrace Far Red DDAO-SEBold recommended fluorophore.7www.invitrogen.com

Flow CytometryFluorophore selection guideInstrument: BD FACScan Flow fault filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488FL1 Green530/30515–545Alexa Fluor 488Click-iT EdU Alexa Fluor 488FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1CFSEGFPJC-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1Argon488FL2 Yellow585/42564–606RPEPIAlexa Fluor 546Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalSNARF (low pH)JC-1 or DiOC2(3)pHrodo dyeArgon488FL3 Red650LP 650RPE-Alexa Fluor 700Qdot 655 nanocrystal7-AADQdot 705 nanocrystalSNARF (high pH)Qdot 800 nanocrystalJC-1 or DiOC2(3)RPE-Cy 5.5LIVE/DEAD Fixable Red Dead Cell StainTRI-COLOR RPE-Cy 7RPE-Alexa Fluor 750PerCPBold recommended fluorophore.8PI

Fluorophore selection guideInstrument: BD FACSCanto lt filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488Green530/30515–545Alexa Fluor 488Click-iT EdU Alexa Fluor 488FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1CFSEGFPJC-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1Argon488Yellow585/42564–606RPEPIAlexa Fluor 546Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalSNARF (low pH)JC-1 or DiOC2(3)pHrodo dyeArgon488Red670LP 670RPE-Alexa Fluor 700PI(695/40)*7-AADRPE-Cy 5.5LIVE/DEAD Fixable Red Dead Cell StainTRI-COLOR SNARF (high pH)PerCPQdot 705 nanocrystalArgon488Infrared780/60750–810RPE-Cy 7RPE-Alexa Fluor 750(787/42)*Qdot 800 nanocrystalBold recommended fluorophore. * Recommended filter.continued9www.invitrogen.com