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 |
MW (Daltons, Da) |
Excitation laser (nm) |
Peak Emission (nm) |
Intensity (1=lowest, 5=highest) |
Uses |
| 11
|
FITC (Fluorescein isothiocyanate)
|
389
|
488
|
518
|
3
|
Detected in FL1 channel on most instruments; also a good choice for fluorescence microscopy
|
| 53
|
Alexa Fluor® 488
|
643
|
488
|
519
|
3
|
Detected in FL1 channel on most instruments; photostable; an excellent choice for fluorescence microscopy
|
| 12
|
R-PE
(R-Phycoerythrin)
|
240K
|
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
|
243K
|
488
|
615
|
3
|
Detected in FL3 on single laser instruments
|
| -
|
PE-Alexa Fluor® 610
|
242K
|
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
|
242K
|
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
|
242K
|
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
|
35K
|
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.
|
| 25
|
PE-Cy7
|
242K
|
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
|
1270
|
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)
|
668
|
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 |
MW (Daltons, Da) |
Excitation laser (nm) |
Peak Emission (nm) |
Intensity (1=lowest, 5=highest) |
Uses |
| 17
|
Allophycocyanin (APC)
|
104K
|
633
|
660
|
4
|
Detected in FL4 on dual laser instruments; can be used interchangeably with Cy5 or Alexa Fluor® 647
|
| 51
|
Alexa Fluor® 647
|
1250
|
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
|
1500
|
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
|
106K
|
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
|
1400
|
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.
|
| 10
|
APC-Cy7
|
106K
|
633
|
760
|
2
|
Detected in appropriate channel based on filter set arrangement of the instrument; tandem dye, resonance energy transfer from APC molecule to cyanine dye Cy7, dim tandem dye
|
| 27
|
APC-Alexa Fluor® 750
|
106K
|
633
|
775
|
2
|
Detected in appropriate channel based on filter set arrangement of the instrument; more photostable then APC-Cy7; APC-Cy7 & APC-Alexa Fluor® 750 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 |
MW (Daltons, Da) |
Excitation laser (nm) |
Peak Emission (nm) |
Intensity (1=lowest, 5=highest) |
Uses |
| 52
|
Alexa Fluor® 405
|
1028
|
405
|
421
|
1
|
Extremely dim dye excited by the violet laser; suitable for only the brightest antigens
|
| 57
|
Pacific Blue®
|
339
|
405
|
455
|
2
|
Excited by the violet laser; Pacific Blue® can be used interchangeably with Alexa Fluor® 405, which is typically a dimmer fluorochrome; please make sure to use this conjugate for your brightest antigen/proteins of interest
|
| -
|
Alexa Fluor® 430
|
702
|
405
|
541
|
1
|
Can be used as second dye excited by the violet laser
|
| -
|
Pacific Orange®
|
|
405
|
551
|
1
|
Although dim and better suited to densely expressed targets, due to its 551nm emission wavelength the Pacific Orange® dye works well with Pacific Blue® for multicolor analysis with the violet or UV light source.
|
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.
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