Multicolor Flow Cytometry



Red Laser Overview

Fluorochromes

 

APC

Allophycocyanin (APC) (Ex Max 650 nm/Em Max 660 nm) is an accessory photosynthetic pigment found in blue-green algae. Its molecular weight is approximately 105 kDa. APC has six phycocyanobilin chromophores per molecule, which make it a very bright fluorochrome that is highly suitable for flow cytometry applications. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.

Filter: 660/20
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)

 

Alexa Fluor® 647

Alexa Fluor® 647 (Ex Max 650 nm/Em Max 668 nm) conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.

Filter: 660/20
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)

 

BD Horizon™ APC-R700

BD Horizon™ APC-R700 (Ex Max 652 nm/Em Max 704 nm) is a tandem fluorochrome that combines APC with R700, a proprietary organic dye. This dye has been developed exclusively by BD Biosciences as a brighter alternative to Alexa Fluor® 700. Due to similar excitation and emission properties, APC-R700 and Alexa Fluor® 700 cannot be used simultaneously.

Filter: 730/45
Relative brightness: Bright (on a scale of Dim, Moderate, Bright, and Brightest)
Sample Data:

Cells stained with APC-R700 show a large spread between positive and negative populations similar to cells stained with PE
Cells stained with APC-R700 show a large spread between positive and negative populations similar to cells stained with PE
Lysed whole blood stained with BD Horizon APC-R700, PE, and Alexa Fluor® 700 conjugated to CD56 (NCAM16.2) on a BD FACSCanto system, 10-color configuration.

 
 

Alexa Fluor® 700

Alexa Fluor® 700 (Ex Max 696 nm/Em Max 719 nm) is a far-red dye that can be excited with a 633–640-nm laser. This enables multicolor analysis in conjunction with APC and Alexa Fluor® 647, or APC-H7 and APC-Cy™7 reagents.

Filter: 730/45
Relative brightness: Dim (on a scale of Dim, Moderate, Bright, and Brightest)

 

APC-Cy™7

APC-Cy7 (Ex Max 650 nm/Em Max 785 nm) is a tandem fluorochrome that combines APC and a cyanine dye (Cy7). Special precautions must be taken with APC-Cy7 conjugates, and cells stained with them, to protect the fluorochrome from long-term exposure to light. Some APC-Cy7 conjugates show changes in their emission spectra with prolonged exposure to paraformaldehyde. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage. Due to nearly identical excitation and emission properties but different spillover characteristics, APC-Cy7 and APC-H7 cannot be used simultaneously.

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

 

BD™ APC-H7

BD APC-H7 (Ex Max 650 nm/Em Max 785 nm) is an APC-cyanine tandem fluorochrome, which uses an analog of Cy7 and has similar spectral properties to APC-Cy7. APC-H7 conjugates provide greater stability in light and paraformaldehyde fixatives and have less spillover into the APC channel than APC-Cy7 conjugates. APC-H7 conjugates are typically 75% as bright as equivalent APC-Cy7 conjugates. Due to nearly identical excitation and emission properties but different spillover characteristics, APC-Cy7 and APC-H7 cannot be used simultaneously.

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

Red Laser - BD APC-H7

APC-Cy7: US Patent 5,714,386


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

Pacific Blue™ and Pacific Orange™ are trademarks, and Alexa Fluor® and Texas Red® are registered 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.