多色流式細胞儀



Blue Laser Overview

螢光物質

 

FITC

FITC (Ex-Max 494 nm/Em-Max 520 nm), fluorescein isothiocyanate, is a fluorochrome with a molecular weight of 389 Da. FITC is sensitive to pH changes and photobleaching. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. FITC is relatively dim and should be reserved for highly expressed markers whenever possible.

Filter: 530/30
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)

 

Alexa Fluor® 488

Alexa Fluor 488 (Ex-Max 495 nm/Em-Max 519 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of FITC. However, Alexa Fluor 488 tends to be brighter and more optimal for intracellular applications. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor 488 cannot be used simultaneously. Alexa Fluor 488 exhibits extraordinary photostability, which makes it highly suitable for fluorescence microscopy.

Filter: 530/30
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)

 

PE

PE (Ex-Max 496 nm/Em-Max 578 nm), R-phycoerythrin (PE), is an accessory photosynthetic pigment found in red algae. It exists in vitro as a 240-kDa protein with 23 phycoerythrobilin chromophores per molecule. This makes PE the brightest fluorochrome for flow cytometry applications, but its photobleaching properties make it unsuitable for fluorescence microscopy.

Filter: 575/26
Relative brightness: Bright (on a scale from Dim, Moderate, Bright, and Brightest)

 

BD Horizon™ PE-CF594

BD Horizon PE-CF594 (Ex-Max 496 nm/Em-Max 612 nm) is a tandem conjugate, developed exclusively by BD Biosciences, that combines PE and CF594. PE-CF594 is a brighter alternative to PE-Texas Red®, with improved spectral characteristics. PE-CF594 reagents exhibit very consistent spillover values lot-to-lot, making them an ideal choice for the PE-Texas Red® detector (610/20 nm).

Filter: 610/20
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)
Sample data:

Staining comparison of BD Horizon PE-CF594, ECD, and PE-Alexa Fluor® 610
Staining comparison of BD Horizon PE-CF594, ECD, and PE-Alexa Fluor® 610

A. CD3: Lysed whole blood stained with human CD3 conjugated to BD Horizon PE-CF594, PE-Texas Red®, ECD, or PE-Alexa Fluor® 610, run on a BD™ LSR II system (using a 610/20-nm filter and the 488-nm laser). All conjugates were run at the manufacturer’s recommended concentrations.

B. CD4: Lysed whole blood stained with human CD4 conjugated to BD Horizon PE-CF594, ECD (PE-Texas Red®, Beckman Coulter), or PE-Alexa Fluor® 610, run on a BD LSR II system (using a 610/20-nm filter and the 488-nm laser). All conjugates were run at the manufacturer’s recommended concentrations.

Example of BD Horizon PE-CF594 staining using various protocols
Example of BD Horizon PE-CF594 staining using various protocols
Histogram overlays of human CD3 or CD19 staining using various staining protocols: live-cell stain, cell staining post-fixation/permeabilization, or cell staining prior to fixation/permeabilization.
Example data from a 10-color TBMNK panel
Example data from a 10-color TBMNK panel
Example data of BD Horizon PE-CF594 used in a 10-color panel to phenotype T cells, B cells, monocytes, and NK cells. CD8 vs CD57 was used to look at CD8 terminal differentiation of the NK cells. Peripheral blood mononuclear cells (PBMCs) were stained with CD16 BD Horizon PE-CF594, CD3 BD Horizon™ V450, CD45 BD Horizon™ V500, CD57 FITC, CD19 PE, CD4 PerCP-Cy™5.5, CD14 PE-Cy™7, CD56 APC, CD20 Alexa Fluor® 700, and CD8 APC-H7. This panel was run on a BD LSRFortessa™ system with violet (405-nm), blue (488-nm), and red (640-nm) lasers.
Example data from a 9-color memory/effector T-cell panel
Example data from a 9-color memory/effector T-cell panel
Example data of BD Horizon PE-CF594 used in a 9-color panel to phenotype various T-cell populations, including terminal effector memory (TEM), effector memory (EM), and central memory (CM). Lysed whole blood was stained with human CD27 BD Horizon PE-CF594, CD62L BD Horizon V450, CD4 BD Horizon V500, CD3 FITC, CCR7/CD197 PE, CD28 PerCP-Cy5.5, CD127 APC, CD45RA Alexa Fluor® 700, and CD8 APC-H7. This panel was run on a BD LSRFortessa system with violet (405-nm), blue (488-nm), yellow-green (561-nm), and red (640-nm) lasers. The PE and BD Horizon PE-CF594 reagents were excited by the 561-nm laser.

 
 

PE-Cy™5

PE-Cy5 (Ex-Max 496 nm/Em-Max 667 nm) is a tandem conjugate that combines phycoerythrin and a cyanine dye. Because of its broad absorption range and the fact that its emission spectra are equivalent to APC, PE-Cy5 is not recommended for simultaneous use with APC. The Cy5 molecule has been shown to exhibit nonspecific binding to Fc receptors, which is most apparent on monocyte populations. PE-Cy5 is not recommended for fluorescence microscopy because it is subject to photobleaching.

Filter: 670/14
Relative brightness: Brightest (on a scale from Dim, Moderate, Bright, and Brightest)

 

PerCP

PerCP (Ex-Max 482 nm/Em-Max 678 nm) is a component of the photosynthetic apparatus found in the dinoflagellate Glenodinium. PerCP is a protein complex with a molecular weight of approximately 35 kDa. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on flow cytometers with high-power lasers (>25 mW).

Filter: 695/40
Relative brightness: Dim (on a scale from Dim, Moderate, Bright, and Brightest)

 

PerCP-Cy™5.5

PerCP-Cy5.5 (Ex-Max 482 nm/Em-Max 695 nm) is a tandem conjugate that combines PerCP with a cyanine dye. PerCP-Cy5.5 is not subject to photobeaching like PerCP and can be used with stream-in-air flow cytometers. It has less Fc receptor-mediated nonspecific staining than PE-Cy5. Additionally, the PerCP-Cy5.5 tandem conjugate is not as susceptible to fixative or light instability as APC-Cy7 and PE-Cy7.

Filter: 695/40
Relative brightness: Moderate (on a scale from Dim, Moderate, Bright, and Brightest)

 

PE-Cy™7

PE-Cy7 (Ex-Max 496 nm/Em-Max 785 nm) is a tandem fluorochrome that combines PE and a cyanine dye. PE-Cy7 conjugated reagents are as bright as PE conjugates. PE-Cy7 is particularly sensitive to photo-induced degradation, resulting in loss of fluorescence and changes in fluorescence spillover. Extreme caution must be taken to avoid light exposure and prolonged exposure to paraformaldehyde fixative. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage.

Filter: 780/60
Relative brightness: Bright (on a scale from Dim, Moderate, Bright, and Brightest)


 


For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Alexa Fluor® is a registered trademark, Pacific Orange™ and Pacific Blue™ are trademarks of Life Technologies Corporation.

Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.

CF™ is a trademark of Biotium, Inc.