Lane Lab

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)

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Combinations of Dyes and Fluorochromes:

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.

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Fluorochromes for LSC Applications:
Nuclear Dyes Fluorochromes
Specialty Dyes Flouresecnt Proteins (GFP etc)

*Tables Adapted from CompuCyte

Nuclear Dyes* (a short List);

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)

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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

-NIH Image Software programs


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