Fluorescent Dyes for Flow Cytometric Analysis
Introduction
Fluorescent dyes absorb light at certain wavelengths and in turn emit
their fluorescence energy at a higher wavelength. Each dye has a
distinct emission spectrum, which can be exploited for multicolor
analysis. eBioscience antibodies are available conjugated to a wide
variety of fluorochromes.
The
tables below lists the properties of the most commonly used
fluorochromes in flow cytometry. The eBioscience product number is the
prefix used in our catalog numbering system to indicate the
corresponding formats of our antibodies. We are continually developing
new fluorochrome conjugated antibodies for both flow cytometry and
microscopy. Please feel free to contact us at tech@ebioscience.com for
more information!
Table 1: Fluorescent Dyes for Flow Cytometric Analysis - 488nm Excitation
|
Fluorescent Dyes for Flow Cytometric Analysis - 488nm Excitation
|
| eBioscience Product Prefix # |
Name |
Excitation laser (nm) |
Peak Emission (nm) |
Intensity (1=lowest, 5=highest) |
Uses |
| 11
|
FITC (Fluorescein isothiocyanate)
|
488
|
518
|
3
|
Detected in FL1 channel on most instruments; also a good choice for fluorescence microscopy
|
| 53
|
Alexa Fluor® 488
|
488
|
519
|
3
|
Detected in FL1 channel on most instruments; photostable; an excellent choice for fluorescence microscopy
|
| 12
|
R-PE
(R-Phycoerythrin)
|
488
|
575
|
5
|
Detected in FL2 channel on most
instruments; very high quantum yield (ideal for detection of low
density antigens); subject to photobleaching.
|
| -
|
PE-Texas Red
|
488
|
615
|
3
|
Detected in FL3 on single laser instruments
|
| -
|
PE-Alexa Fluor® 610
|
488
|
628
|
3
|
This alternative to PE-Texas Red has less
overlap with PE due to its 628nm emission wavelength and is less
subject to Fc mediated binding than PE-Cy5.
|
| 15
|
PE-Cy5
|
488
|
670
|
4
|
Detected in FL3 or FL4 on most
instruments; tandem dye, resonance energy transfer from PE molecule to
the cyanine dye Cy5; when used with APC on dual laser machines, needs a
cytometer capable of interlaser compensation. See PE-Cy5.5 for dual
laser instruments
|
| 35
|
PE-Cy5.5
|
488
|
690
|
3
|
Detected in FL3 or FL4 on most
instruments; tandem dye, resonance energy transfer from PE molecule to
cyanine dye Cy5.5; when used with APC on dual laser machines, needs a
cytometer capable of interlaser compensation. Better choice for dual
laser instruments than PE-Cy5.
|
| 45
|
PerCP-Cy5.5
|
488
|
690
|
3
|
Detected in FL3 or FL4 on most
instruments; tandem dye, resonance energy transfer from PerCP molecule
to cyanine dye Cy5.5; when used with APC on dual laser machines, needs
a cytometer capable of inter-laser compensation. Better choice for dual
laser instruments than PE-tandems as a result of its exceptionally
large stokes shift.
|
| 46
|
PerCP-eFluor™ 710
|
488
|
710
|
4
|
Detected in FL3 on most instruments;
tandem dye, resonance energy transfer from PerCP molecule to eFluor™
710; when used with APC on dual laser machines, needs a cytometer
capable of inter-laser compensation. Better choice for dual laser
instruments than PE-tandems as a result of its exceptionally large
stokes shift. 2-3 fold brighter than PerCP-Cy5.5.
|
| 25
|
PE-Cy7
|
488
|
760
|
3
|
Detected in appropriate channel based on
filter set arrangement of the instrument; tandem dye, resonance energy
transfer from PE molecule to cyanine dye Cy7; minimal cross-beam
compensation required when used simultaneously
|
| -
|
7-AAD
|
488
|
647
|
|
Detected in FL3 channel on most
instruments; binds selectively to GC regions of DNA; used as a
viability dye to discriminate between viable and non-viable cells
|
| -
|
Propidium Iodide (PI)
|
488
|
617
|
|
Detected in FL2 and FL3 channels on most
instruments; a DNA intercalating dye; used as a viability dye to
discriminate between viable and nonviable cells
|
Table 2: Fluorescent Dyes for Flow Cytometric Analysis - 633nm Excitation
|
Fluorescent Dyes for Flow Cytometric Analysis - 633nm Excitation
|
| eBioscience Product Prefix # |
Name |
Excitation laser (nm) |
Peak Emission (nm) |
Intensity (1=lowest, 5=highest) |
Uses |
| 17
|
Allophycocyanin (APC)
|
633
|
660
|
4
|
Detected in FL4 on dual laser instruments; can be used interchangeably with Cy5 or Alexa Fluor® 647
|
| 51
|
Alexa Fluor® 647
|
633
|
668
|
4
|
Detected in FL4 on dual laser
instruments; can be used interchangeably with Cy5 or APC; photostable,
making it a great choice for microscopy, in addition to flow cytometry
|
| 19
|
Cy5
|
633
|
670
|
3
|
Detected in FL4 on dual laser instruments (used interchangeably with APC or Alexa Fluor® 647); good choice for intracellular labeling and microscopy
|
| 30
|
APC-Cy5.5
|
633
|
690
|
3
|
Detected in appropriate channel based on
filter set arrangement; tandem dye, resonance energy transfer from APC
molecule to Cy5.5
|
| 56
|
Alexa Fluor® 700
|
633
|
723
|
2
|
Although not optimally excited by the
He-Ne laser, it does produce a bright signal that can be used as a
third dye off this laser; please note that emission wavelength requires
a distinct set of filters; most instruments will require a 685 LP
mirror and 710/20 filter.
|
| 47
|
APC-eFluorTM 780
|
633
|
780
|
2
|
Appropriate channel for detection based
on filter set arrangement of the instrument; more photostable then
APC-Cy7; tandem dye, resonance energy transfer from APC molecule to
eFluorTM 780, APC-Cy7, APC-Alexa Fluor® 750 and APC-eFluorTM 780 may be used interchangeably.
|
| 27
|
APC-Alexa Fluor® 750
|
633
|
775
|
2
|
Appropriate channel for detection based
on filter set arrangement of the instrument; more photostable then
APC-Cy7; tandem dye, resonance energy transfer from APC molecule to
Alexa Fluor® 750, dim tandem dye. APC-Cy7, APC-Alexa Fluor® 750 and APC-eFluorTM 780 may be used interchangeably.
|
Table 3: Fluorescent Dyes for Flow Cytometric Analysis - 405nm Excitation
|
Fluorescent Dyes for Flow Cytometric Analysis - 405nm Excitation
|
| eBioscience Product Prefix # |
Name |
Excitation laser (nm) |
Peak Emission (nm) |
Intensity (1=lowest, 5=highest) |
Uses |
| 93
|
eFluorTM 605NC
|
405
|
605
|
2
|
Excited by the violet laser; eFluorTM 605NC can be used in combination with eFluorTM 450, please make sure to use this conjugate for your brightest antigen/proteins of interest
|
| 94
|
eFluorTM 625NC
|
405
|
625
|
2
|
Excited by the violet laser; eFluorTM 625NC can be used in combination with eFluorTM 450, please make sure to use this conjugate for your brightest antigen/proteins of interest
|
| 95
|
eFluorTM 650NC
|
405
|
650
|
3
|
Excited by the violet laser; eFluorTM 650NC can be used in combination with eFluorTM 450 and eFluorTM 605NC, please make sure to use this conjugate for your brightest antigen/proteins of interest.
|
| 48
|
eFluorTM 450
|
405
|
450
|
2
|
Excited by the violet laser; eFluorTM 450 can be used interchangeably with Pacific Blue®, can be used in combination with eFluorTM 605NC or eFluorTM 625NC; please make sure to use this conjugate for your brightest antigen/proteins of interest
|
| 52
|
Alexa Fluor® 405
|
405
|
421
|
1
|
Extremely dim dye excited by the violet laser; suitable for only the brightest antigens
|
| 57
|
Pacific Blue®
|
405
|
455
|
2
|
Excited by the violet laser; Pacific Blue® can be used interchangeably with eFluorTM 450, typically a dimmer fluorochrome; please make sure to use this conjugate for your brightest antigen/proteins of interest
|
| -
|
Alexa Fluor® 430
|
405
|
541
|
1
|
Can be used as second dye excited by the violet laser
|
| -
|
Pacific Orange®
|
405
|
551
|
1
|
Dim and only suitable for densely expressed targets, due to its 551nm emission wavelength the Pacific Orange® dye can be used with Pacific Blue® for multicolor analysis with the violet or UV light source, however eFluorTM 605NC is a better alternative
|
Cy-ChromeTM is a registered trademark of Becton, Dickinson and Company. Tri-ColorTM is a registered trademark of Caltag Laboratories. Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc., for research use only.
|
