Core Facilities
USF-Health College of Medicine

 
      


Fluorescence

In Flow Cytometry we measure light, frequently fluorescent light.  If you are not familiar with issues concerning fluorescence and how those relate to biological work, there are some good tutorials on the topic on the Invitrogene website here.

The Invitrogen tutorial gives a good idea about the nature of fluorescent dyes.  There are two more items worth mentioning.  One is compensation, which is well covered in tutorials under the "Flow Cytometry" tab.  The other one is brightness of fluorochromes, in terms of which one to pick for which parameter you are interested in. In the preparation and planning of an experiment, the combination of fluorochromes per acquisition is critical especially when investigating cells occurring at low frequency or when utilizing multi-parameter flow cytometry.

Two points are of particular importance when deciding which fluorochromes to use. First, not all fluorochromes are made equal; some are brighter than others e.g. PE and APC both emit a higher wavelength when excited and so stain with a higher relative fluorescent intensity. Second, the final decision of which fluorochrome to use maybe dependent upon; the number of lasers in the flow cytometer, confidence in setting the compensation parameter for the second laser and/or which antibody you can find at the time! A good method is to generally stain the least frequent cells with the brightest fluorochrome and visa versa. Brightness depends not only on the inherent qualities of the fruorochrome, but also on the detectors/filter sets of the equipment that will detect the light, so the same fluorochromes might have different order using different machines. A general guideline to the relative emission wavelengths of each fluorochrome is shown below.

Brightest  PE, PE-Cy7, PE-Cy5, APC > APC-Cy7>Alexa Fluor 647, Alexa Fluor 700 >FITC, Pacific Blue, Alexa Fluor 488 Least bright

So, there you are.  The only commercially available antibody (and it is a great antibody) conjugate to your rare population is FITC instead of APC.  Is there anythithing that can be done to help this situation, you ask.  Milteny Biotec thinks, yes. FITCenhancer.pdf  If your staining of APC or PE is not strong enough, they have a kit to enhance those, too.

 

Regarding the brightness there are more issues that make the question tricky.  What if dye A is brighter then dye B if excited at the optimal excitation wavelength, but the one in the flow cytometer is closer to the optimum for B than for A?  You are using the dyes for flow and for immunocytochemistry, A is brighter than B, but B is more photo-stabile, and you are likely to spend some time scanning the slide before taking a picture.  Which one will be brighter at the time of talking a picture?  You can read more on this topic by Mario Roederer or the BD 2006 tech resource page BDISTechResources2006.pdf.

 

Another important question is which fluorochromes can be used with our equipment.  The following tables should be a good guide in this regard for the basic configuration of the Canto II and the LSR II.  The most frequently used fluorochromes are highlighted, the ones that can be detected with the particular detector in a sub-optimal back up scenario are in parenthesis.

 

 

Canto II 488 LASER:

 
Detector LP Band Pass Fluors
A 735 780/60

PE-CY7, CyQuantDNA

B 655 670/LP

7AAD, Alexa647-PE, PE-CY5, PerCP, PerCP-Cy5.5, PE-Cy5.5, (PI)

C 610 none

No PMT

D 556 585/42

Alexa532, DsRed, CytoxOr, PE, PI, EB,(Alexa430, OsRed)

E 502 530/30

eYFP, FITC, Alexa 430,  Alexa 488,  Alexa 532, BODIPY, Calcein, DCF, (GFP*), Fluo-3, Fluoro-emerald, Na-Green, OregonGreen, PicoGreen, Rho110, Rho123, CytoxGr, To-Pro-1, ZsGreen

F none 488/10

SSC

G - - -
H - - -

 

Canto II 633 LASER

 
Detector LP Band Pass Fluors
A 735 780/60

APC-CY7, Alexa750

B 685 -

No PMT

C - 660/20

APC, Alexa 633, Alexa 647, CPC, Cy5, To-PRO-3

 

LSR II 488 Blue LASER:

 
Detector LP Band Pass Fluors
A 755 780/60

PE-CY7

B 690 710/50

PerCp-CY5.5, PE-CY5.5, FuraRedLo

C 675 688/35

7AAD, Alexa647-PE, PE-CY5, PerCp-CY5.5, PE-CY5.5, Cychrome, PerCP, (PI)

D 600 610/20

(7AAD), Alexa610-PE, DsRed, EB, (NileRed), PI, PE-Texas Red

E 550 575/26

DsRed, PE

F 505 530/30

Alexa488, Alexa532, PODIPY, Calcein, eYFP, eGFP, FITC, Fluo3, Fluo-emerald, NaGreen, OregonGr, PicoGreen, Rho110, Rho123, SytoxGr, ToPro1, ZsGreen

G - 488/10

SSC

H - - -



LSR II 404 Violet LASER:

 
Detector LP Band Pass Fluors
A 595 605/40

Qdot605

B 505 525/50

Amcyan, CascadeY, eCFP, live/dead Fixed Aqua, Qdot525, Qdot545

C - 450/40

Alexa 405, Alexa430, Cascade Blue, live/dead fixable Violet, Pacific blue, DAPI, Hoechst**, eCFP

LSR II 633 LASER

 

Detector

LP

Band Pass

Fluors

A

755

780/60

APC-CY7, Alexa750

B

685

735/45

Alexa 680, Alexa 700

C

-

660/20

APC, Alexa 633, Alexa 647, CPC, Cy5, To-PRO-3

Commonly Used Fluorescent Proteins

Protein
Names

Reference or Source

Spectral Properties

Oligomeric State

 
   

Excitation
nm

Emission
nm

Brightness relative to eGFP

 

Argon laser, nm

Blue Fluorescent Proteins

Azurite

Mena et al., Nat. Biotechnol., 2006, 24, 1569

383

447

0.43

monomer

404

eBFP

Yang et al., J. Biol. Chem., 1998, 273, 8212

380

440

0.27

monomer

404

SuperGlo BFP

www.qbiogene.com

387

450

n/a

monomer

404

Cyan Fluorescent Proteins

eCFP

www.clontech.com

439

476

0.39

monomer

404

Cerulean

Rizzo et al., Nat. Biotechnol., 2004, 22, 445

433 475 0.79 monomer 404

CyPet

Nguyen et al., Nat. Biotechnol., 2005, 23, 355

435

477

0.53 monomer 404

Midoriishi Cyan

www.mblintl.com

472

495

0.73 dimer 488

Yellow Fluorescent Proteins

eYFP

www.clontech.com

514 527 1.51

monomer

488

Topaz

Cubitt et al., Methods Cell. Biol., 1999, 58, 19

514

527

1.69

monomer

488

Venus

Nagai et al., Nat. Biotechnol., 2002, 20, 87

515 528 1.56

monomer

488

mCitrine

Griesbeck et al., J. Biol. Chem., 2001, 276, 29188

516 529 1.74

monomer

488

YPet

Nguyen et al., Nat. Biotechnol., 2005, 23, 355

517

530

2.38

monomer

488

TurboYFP

www.evrogen.com

525 538 1.65 dimer 488

Red Fluorescent Proteins

DsRed

www.clontech.com

588 583 1.76

tetramer

488

DsRed-
Express (T1)

www.clontech.com

555

584

0.58

tetramer

488

DsRed-
Monomer

www.clontech.com

556 586 0.10

monomer

488

mKeima

Kogure et al., Nat. Biotechnol., 2006, 24, 577

440 620 0.12

monomer

404

Photo-Switchable Fluorescent Proteins

PA-GFP

 

before activation     

400

515

0.08

monomer

n/a

after activation     

504 517 0.42 488

PS-CFP2

 
before activation     400 470 0.26 monomer n/a
after activation     490

511

0.33 488

Dronpa

           
before activation     n/a n/a 0.01 monomer n/a
after activation     503

518

2.45 488

Kaede

   

 

     
before activation     508 518 2.64 monomer 488
after activation     572

580

0.60 n/a

mEosFP

           
before activation     505 516 1.30 monomer 488
after activation     569

581

0.70 n/a

Dendra2

           
before activation     490 507 0.45 monomer 488
after activation     553

573

0.39 n/a
 

 

             
While the list above is quite large, but by all means is not a definitive one.  If you have something else on your mind, discuss it with the Flow Lab.

If you are looking for the right fluorochrome combination for your experiment the "Fluorochrome Spectra" link is here to help you.