Fluorophore selection guide for flow cytometry

1 Flow CytometryFluorophore selection guide for flow cytometryDye choices for your instrument made selection guide for flow cytometryDyes for the 405 nm and 407 nm lasers .. 2 Dyes for the 488 nm laser.. 3 Dyes for the 635 nm laser.. 5 Accuri C6 Flow Cytometer System .. 6BD FACSC alibur System .. 7BD FACScan Flow Cytometer.. 8BD FACSC anto System.. 9BD FACSC anto II System, 2-laser option .. 11BD FACSC anto II System, 3-laser option .. 13BD LSR Flow Cytometer .. 15BD LSR II Flow Cytometer .. 17BD FACSAria Cell Sorter .. 19BD FACSA rray Bioanalyzer System.. 21BD FACSV antage SE Cell Sorter.. 22 Beckman Coulter Cytomics FC 500 Flow Cytometer .. 25 Beckman Coulter EPICS XL Flow Cytometer.. 27 Beckman Coulter Cell Lab Quanta SC .. 28 Beckman Coulter EPICS ALTRA Cell Sorter .. 30 Beckman Coulter CyAn ADP Analyzer .. 33 Beckman Coulter MoFlo XDP Cell Sorter .. 35 Guava Personal Cell Analysis (PCA) System .. 38 Guava EasyCyte Mini System.

2 39 Guava EasyCyte Plus System .. 40 Index.. 412 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 fluorophores Alexa 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 excitation Pacific 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 ( , 548/42 or 575/26) Qdot 525nanocrystalQdot 565nanocrystalQdot 605nanocrystalQdot 655nanocrystalQdot 705nanocrystalQdot 800 nanocrystalAllows 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) conjugates 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 cytometry RPE (R-Phycoerythrin)

3 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 photobleaching RPE-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 instruments RPE-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 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 instrument TRI-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 FACSC alibur instruments Measured in FL4 channel on Beckman Coulter EPICS XL instrument Can demonstrate monocyte binding in some circumstances continued4 Flow CytometryFluorophore selection guideDyes for the 488 nm laser, continuedRPE-Alexa Fluor 700 Fluorochrome of choice for BD FACSC alibur 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 FACSC alibur or BD FACScan instrument Bright alternative to RPE-Cy 5 (TRI-COLOR , CyChrome, PC5), PerCP, and RPE-Cy Together with RPE-Alexa Fluor 610 dye, constitutes the optimal four-color combination using the 488 nm laser line RPE- Cy 5.

4 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 FACSC alibur instruments Measured in FL4 on Beckman Coulter EPICS XL instrument RPE-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 channel 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 5 Alexa 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.

5 Small-molecule direct conjugate alternative to the tandem APC-Cy Minimal compensation against APC Requires significant compensation against RPE-Alexa Fluor 700 and RPE-Cy APC-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 7 Fluorophore selection guideDyes for the 635 nm laser6 Flow CytometryLaserExcitation laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488FL1 Green530/30515 5 45 Alexa Fluor 488 Click-iT EdU Alexa Fluor 488 FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1 CFSEGFPJ C-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123 LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1 Argon488FL2 Yellow585/4 0565 605 RPEPIA lexa Fluor 546 Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalJ C-1 or DiOC2(3)SNARF (low pH)pHrodo dyeArgon488FL3 Red675LP 675 RPE-Alexa Fluor 700 PIQdot 705 nanocrystal7-AADQdot 800 nanocrystalSNARF (high pH)RPE- Cy C-1 or DiOC2(3)TRI-COLOR LIVE/DEAD Fixable Red Dead Cell StainRPE- Cy 7 RPE-Alexa Fluor 750 PerCPHe Ne640FL4 Red675/24663 687 APCC lick-iT EdU Alexa Fluor 647 Alexa Fluor 647 LIVE/DEAD Fixable Far Red Dead Cell StainQdot 655 nanocrystalTO-PRO -3 SYTOX RedMitoProbe DiIC1(5)CellTrace Far Red DDAO-SEBold = recommended selection guideInstrument.

6 Accuri C6 Flow Cytometer laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488FL1 Green530/30515 5 45 Alexa Fluor 488 Click-iT EdU Alexa Fluor 488 FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1 CFSEGFPJ C-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123 LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1 Argon488FL2 Yellow585/42564 606 RPEPIA lexa Fluor 546 Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalJ C-1 or DiOC2(3)SNARF (low pH)pHrodo dyeArgon488FL3 Red670LP 670 RPE-Alexa Fluor 700 PIQdot 705 nanocrystal7-AADQdot 800 nanocrystalSNARF (high pH)RPE- Cy C-1 or DiOC2(3)TRI-COLOR LIVE/DEAD Fixable Red Dead Cell StainRPE- Cy 7 RPE-Alexa Fluor 750 PerCPHe Ne635FL4 Red6 61/12656 667 APCC lick-iT EdU Alexa Fluor 647 Alexa Fluor 647 LIVE/DEAD Fixable Far Red Dead Cell StainQdot 655 nanocrystalTO-PRO -3 SYTOX RedMitoProbe DiIC1(5)CellTrace Far Red DDAO-SEBold = recommended selection guideInstrument: BD FACSC alibur System8 Flow CytometryFluorophore selection guideInstrument.

7 BD FACScan Flow CytometerLaserExcitation laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488FL1 Green530/30515 5 45 Alexa Fluor 488 Click-iT EdU Alexa Fluor 488 FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1 CFSEGFPJ C-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123 LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1 Argon488FL2 Yellow585/42564 606 RPEPIA lexa Fluor 546 Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalSNARF (low pH)J C-1 or DiOC2(3)pHrodo dyeArgon488FL3 Red650LP 650 RPE-Alexa Fluor 700 PIQdot 655 nanocrystal7-AADQdot 705 nanocrystalSNARF (high pH)Qdot 800 nanocrystalJ C-1 or DiOC2(3)RPE- Cy Fixable Red Dead Cell StainTRI-COLOR RPE- Cy 7 RPE-Alexa Fluor 750 PerCPBold = recommended laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488 Green530/30515 5 45 Alexa Fluor 488 Click-iT EdU Alexa Fluor 488 FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1 CFSEGFPJ C-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123 LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1 Argon488 Yellow585/42564 606 RPEPIA lexa Fluor 546 Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalSNARF (low pH)J C-1 or DiOC2(3)pHrodo dyeArgon488 Red670LP 670 RPE-Alexa Fluor 700 PI(695/40)*7-AADRPE- Cy Fixable Red Dead Cell StainTRI-COLOR SNARF (high pH)PerCPQdot 705 nanocrystalArgon488 Infrared780/60750 810 RPE- Cy 7 RPE-Alexa Fluor 750 ( 787/42)*Qdot 800 nanocrystalBold = recommended Fluorophore .

8 * Recommended selection guideInstrument: BD FACSC anto Systemcontinued10 Flow CytometryFluorophore selection guideInstrument: BD FACSC anto System, continuedLaserExcitation laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesHe Ne633 Red660/20650 670 APCC lick-iT EdU Alexa Fluor 647 Alexa Fluor 647 LIVE/DEAD Fixable Far Red Dead Cell StainQdot 655 nanocrystalTO-PRO -3 SYTOX RedMitoProbe DiIC1(5)CellTrace Far Red DDAO-SEHe Ne633 Infrared780/60750 810 APC-Alexa Fluor 750 LIVE/DEAD Fixable Near-IR Dead Cell Stain(787/42)*A P C- Cy 7 Qdot 800 nanocrystalBold = recommended Fluorophore . * Recommended laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488 Green530/30515 5 45 Alexa Fluor 488 Click-iT EdU Alexa Fluor 488 FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1 CFSEGFPJ C-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123 LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1 Argon488 Yellow585/42566 606 RPEPIA lexa Fluor 546 Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalSNARF (low pH)J C-1 or DiOC2(3)pHrodo dyeArgon488 Orange616/ 2 3604 628 RPE-Alexa Fluor 610 PIRPE-Texas Red J C-1 or DiOC2(3)Qdot 705 nanocrystalLIVE/DEAD Fixable Red Dead Cell StainArgon488 Red670LP 670 RPE-Alexa Fluor 700 PI(695/40)*7-AADRPE- Cy Fixable Red Dead Cell StainTRI-COLOR SNARF (high pH)PerCPQdot 705 nanocrystalArgon488 Infrared780/60750 810 RPE- Cy 7 RPE-Alexa Fluor 750 ( 787/42)*Qdot 800 nanocrystalBold = recommended Fluorophore .

9 * Recommended selection guideInstrument: BD FACSC anto II System, 2-laser optioncontinued12 Flow CytometryFluorophore selection guideInstrument: BD FACSC anto II System, 2-laser option, continuedLaserExcitation laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesHe Ne633 Red660/20650 670 APCC lick-iT EdU Alexa Fluor 647 Alexa Fluor 647 LIVE/DEAD Fixable Far Red Dead Cell StainQdot 655 nanocrystalTO-PRO -3 SYTOX RedMitoProbe DiIC1(5)CellTrace Far Red DDAO-SEHe Ne633 Far Red712 / 21701 723 Alexa Fluor 700He Ne633 Infrared780/60750 810 APC-Alexa Fluor 750 LIVE/DEAD Fixable Near-IR Dead Cell Stain(787/42)*A P C- Cy 7 Qdot 800 nanocrystalBold = recommended Fluorophore . * Recommended filter. Available on some models. laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesArgon488 Green530/30515 5 45 Alexa Fluor 488 Click-iT EdU Alexa Fluor 488 FluoresceinCalceinQdot 525 nanocrystalFluo-3 or Fluo-4TO-PRO -1 CFSEGFPJ C-1 or DiOC2(3)SYTOX GreenDyeCycle GreenRhodamine 123 LIVE/DEAD Fixable Green Dead Cell StainYO-PRO -1 Argon488 Yellow585/42566 606 RPEPIA lexa Fluor 546 Fura Red Qdot 565 nanocrystalDyeCycle OrangeQdot 585 nanocrystalSNARF (low pH)J C-1 or DiOC2(3)pHrodo dyeArgon488 Red670LP 670 RPE-Alexa Fluor 700 PI(695/40)*7-AADRPE- Cy Fixable Red Dead Cell StainTRI-COLOR SNARF (high pH)PerCPQdot 705 nanocrystalArgon488 Infrared780/60750 810 RPE- Cy 7 RPE-Alexa Fluor 750 ( 787/42)*Qdot 800 nanocrystalBold = recommended Fluorophore .

10 * Recommended selection guideInstrument: BD FACSC anto II System, 3-laser option14 Flow CytometryFluorophore selection guideInstrument: BD FACSC anto II System, 3-laser option, continuedLaserExcitation laserFluorescence channelDefault filterFilter range (nm)FluorophoresOther fluorescent dyesHe Ne633 Red660/20650 670 APCC lick-iT EdU Alexa Fluor 647 Alexa Fluor 647 LIVE/DEAD Fixable Far Red Dead Cell StainQdot 655 nanocrystalTO-PRO -3 SYTOX RedMitoProbe DiIC1(5)CellTrace Far Red DDAO-SEHe Ne633 Infrared780/60750 810 APC-Alexa Fluor 750 LIVE/DEAD Fixable Near-IR Dead Cell Stain(787/42)*A P C- Cy 7 Qdot 800 nanocrystalViolet 405 Blue450/50425 475 Pacific Blue Click-iT EdU Pacific Blue Alexa Fluor 405PO-PRO -1 DyeCycle VioletLIVE/DEAD Fixable Violet Dead Cell StainCalcein VioletViolet 405 Green513/ 2 2502 525 Qdot 525 nanocrystalSYTOX BlueAmCyanLIVE/DEAD Fixable Aqua Dead Cell StainBold = recommended Fluorophore . * Recommended filter.