Lasers and Compatible Dyes/Fluorochomes
| Available Lasers and compatible Dyes | Suitable Combinations of Dyes: |
| Fluorochromes for the LSC | Additional sources of dyes and information |
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The Laser Scanning Cytometer (LSC) has the following lazers:
488nm Argon laser (20mW):
excites: Propidium iodide (PI), DNA stain
FITC PE, PE-Texas Red tandem, TriChrome (PE-Cy5)
Also PerCP, [not recommended due to very short half life]
633nm Helium Neon Laser (20mW):
excites: ToPro-3, DNA stain
Cy5, APC (better than Cy5) APC-Cy7
395nm UV-diode Laser (10 mW ):
excites: DAPI, Hoesht DNA stains
BFP and other blue shifted fluorescent markers
Other Dyes:
The LCS can not adequately excite: Cy3 or Texas red (these require a green HeNe lazer 540nm)
For application of a DNA stain plus 2 or 3 additional probes, here are a couple of options:
Argon Laser. . . . . . . . . . . . . . . . HeNe Laser . . . . . . . . . . . . . . UV diode
1. FITC + PI (DNA) . . . . . . . . . . . . Cy5 or APC . . . . . . . . . . DAPI (DNA) or BFP
2. FITC + PE + Trichrome. . . . . . ToPro3 (DNA). . . . . . . . . . DAPI (DNA) or BFP
Note: with TriChrome type dies, 4-color phenotyping can be done (no DNA stain) using FITC, PE, PerCP, and APC with no problems.
Tandem and Specialty Dies:
-PE-Texas Red belongs to a family of fluorochromes called tandem dyes. The PE is excited by the 488nm Argon laser. The emitted light energy, coming off around 570nm, then excites the Texas Red molecule attached to the PE molecule. This gives an emission of around 610nm, which the LSC collects in channel (PMT)3. Only Coulter and R&D systems makes antibodies conjugated to this tandem.
-TriChrome, or TriColor as it is also called, is actually a PE-Cy5 tandem. This dye will emit at around 670nm and is collected in our 4th channel (PMT). Coulter, BD, Pharmingen, Caltag, DAKO, and others have both human, mouse, and rat antibodies conjugated to this tandem dye.
-PerCP is a unique fluorochrome that is a patented BD dye. It is not always recommended due to very short half life. What makes it unique is its very large stokes shift (difference between the excitation and emission wavelength). PerCP excites with the Argon laser and emits around 680nm. BD has a variety of antibodies conjugated to PerCP. Problem is (and BD will never admit this) this dye has very poor stability and is fairly dim to begin with. It also bleaches very rapidly, making it a poor choice for a system like the LSC. It can be run though if you need it.
Nuclear Dyes Fluorochromes Specialty Dyes Flouresecnt Proteins (GFP etc) *Tables Adapted from CompuCyte
|
Dye
|
Lazer
|
Filters
|
Application:
|
| Acridine Orange |
Ar
|
G, R
|
DNA, RNA discrimination, Lysosome labeling |
| 7-Aminoactinomycin D |
Ar
|
R, LR
|
DNA content, Viability |
| DAPI |
UV
|
||
| Ethidium Bromide |
Ar
|
R, LR
|
dsDNA intercalator, Viability, Chromosome labeling |
| Ethidium Homodimer |
Ar
|
R, LR
|
DNA content, Viability |
| Hoescht |
UV
|
||
| LDS 751 (Styryl-8) |
Ar
|
LR
|
DNA content, Viability, Leucocyte differentiation |
| Propidium Iodide |
Ar, Ne
|
R, LR
|
DNA content, Viability, Chromosome labeling* |
| SYTO(R) 11, 12, 20, 22,16 (m) |
Ar
|
G
|
DNA, RNA content of viable cells |
| SYTO(R) 17, 59, 61 (m) |
Ne
|
LR
|
Cytoplasmic labeling in viable Bacteria and Eukaryotes |
| SYTOX(R) Green (m) |
Ar
|
G
|
Chromosome labeling, Impermeant* |
| Thiazole Blue |
HeNe
|
LR
|
Reticulocyte labeling |
| To-Pro?-3 (m) |
HeNe
|
LR
|
Viability, photosensitive |
Fluorochromes* (a short List);
|
Dye
|
Lazer
|
Filters
|
Application:
|
| Alexa Fluor? 488 (m) |
Ar
|
G
|
Substitute for FITC, better stability and intensity* |
| APC (Allophycocyanine) |
HeNE
|
LR
|
Second most widely used Long Red dye |
| BODIPY(R) FL (m |
Ar
|
G
|
Substitute for FITC, better stability and intensity* |
| BODIPY(R) 630/650 (m) |
HeNE
|
LR
|
Substitute for LR dyes, better stability and intensity |
| CY5 (a) |
HeNE
|
LR
|
Small molecule substitute for APC, TR* |
| CY5.5 (a) |
HeNE
|
LR
|
Small molecule substitute for APC |
| FITC (Fluorescein) |
Ar
|
G
|
Most widely used Green dye* |
| Oregon Green(R) 488 (m) |
Ar
|
G
|
Substitute for FITC, better stability and intensity |
| PE (Phycoerythrin) |
Ar
|
O
|
Most widely used Orange dye (R-form recommended)* |
| PE-APC tandem |
Ar
|
LR
|
|
| PE-CY5 tandem |
Ar
|
LR
|
|
| PerCP |
Ar
|
LR
|
Long Red dye |
| PE-TR (Texas Red(R)) (m) |
Ar, R
|
LR
|
Argon laser excited Texas Red dye |
| Rhodamine Green? (m) |
Ar
|
G
|
Substitute for FITC, better stability and intensity |
| Rhodol Green? (m) |
Ar
|
G
|
Substitute for FITC, better stability and intensity |
Specialty Dyes* (a short List);
|
Dye
|
Lazer
|
Filters
|
Application:
|
| BCECF |
Ar
|
G
|
Cellular membrane potential |
| Calcium Green? (m) |
Ar
|
G
|
Calcium measurements, ratioed with Fura Red |
| Carboxy-DCFDA (m) |
Ar
|
G
|
Reactive oxygen intermediates* |
| Carboxy SNARF(R)-1 AM (m) |
Ar
|
O/R ratio
|
Cellular pH measurements |
| DiIC(N)(5) |
HeNE
|
LR ER
|
Mitochondrial membrane potential* |
| DiOC(N)(3) |
Ar
|
G ER
|
Mitochondrial membrane potential* |
| Fluo-3 (m) |
Ar
|
G
|
Calcium measurements |
| Fura Red? (m) |
Ar
|
R
|
Calcium measurements |
Flouresecnt Proteins and Markers (a short List);
|
Dye
|
Lazer
|
Filters
|
Application:
|
| Green Fluorescent Protein |
Ar
|
G
|
|
| JC-1 |
Ar
|
O, R
|
Mitochondrial membrane potential |
| NBD-C6-Ceramide |
Ar
|
G
|
Golgi apparatus |
*Tables Adapted from CompuCyte (please see their www site for further details and for published referances)
(a) Amersham Life Sciences, Inc., Arlington Heights, IL.
(m) Molecular Probes Inc., Eugene, OR.
Key to Symbols
Ar: Argon 488 nm HeNe: Helium Neon 633 nm
G, O, R, LR: Green, Orange, Red, Long Red optical filter sets in the LSC(R)
Additional Sources of Information:
The following www sites have lots of information on cytometry compatible dyes.
-CompuCyte Manufacturer of the LSC
-Beckton Dickinson (BD) Manufacturer of antisera and fluorescent conjugates
-Pharmingen Now a division of BD
-Caltag Manufacturer of antisera and fluorescent conjugates
-DAKO Manufacturer of antisera and fluorescent conjugates
Molecular Probes Inc Manufacturer of dyes and filter sets
Cytometry Chat groups
-Purdue University Cytometry chat group
Cytometry Software
-CompuSort software used by the LSC
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