Applications > Cell Biology > Antibodies, strepavidin and lectins

Near-IR CF™ Dyes

Simply the brightest and most stable long wavelength dyes

Long Wavelength CF Dyes Ideal for In Vivo Imaging and Other Near-IR Detection

Figure 1: Stability of Cy7, AlexaFluor750 and CF750 dyes. Shown are the absorption spectra of the respective dyes before (black line) and after (gray line) 30 minutes of exposure to sunlight.

Developed by scientists at Biotium, Near-IR CF dyes are a group of fluorescent dyes with absorption and emission wavelengths between 650 and 800 nm. Designed for protein labeling, near-IR CF dyes are significantly brighter and more stable than any other commercial dye of similar wavelengths. Near-IR dyes offer important advantages over traditional visible light dyes. Because cellular or tissue components produce minimal autofluorescence in the near-IR region, near-IR dyes have the potential to offer highly specific and sensitive detection in complex biological systems. Also, because light with wavelength in the near-IR region has strong tissue penetration, near-IR dyes are ideal for in vivo fluorescence imaging, an emerging field that has advanced rapidly in recent years. Futhermore, near-IR dyes are also excellent dyes for in- or on-cell and membrane-based Western assays.

Figure 2: Jurkat cells were stained with isotype or mouse anti-human CD3 antibody followed by goat anti-mouse APC-AlexaFluor750 (Invitrogen) or CF750 using the amount of antibody shown. Flourescence was analyzed using a BD LSR II flow cytometer equipped with a 633 nm laser and 780/60 nm PMT detector. Bars represent the relative fluorescence values of the geometric means.

Near-IR CF dyes are next-generation long wavelength dyes representing a true breakthrough in the field. The current commercial near-IR dyes suffer from problems of limited fluorescence brightness due to excessive dye aggregation and poor stability. As a result of novel molecular engineering by scientists at Biotium, near-IR CF dyes overcome these problems, resulting in several key advantages over other competitive dyes. Near-IR CF dyes are exceptionally bright and stable. For example, our CF750 is so bright, it can be excited at 633 nm (i.e., at the shoulder wavelength of the absorption maximum) but still emits stronger fluorescence at ~770 nm than APC-based tandem dyes, making the dye particularly useful for flow cytometry applications without the spillover and stability challenges encountered with tandem dyes.

In addition, near-IR CF dyes possess a proprietary structural feature that renders the dyes less immunogenic than other competitive dyes. Consequently, a higher number of near-IR CF dyes can be conjugated to a protein for maximal fluorescence, and the resulting protein conjugate can be expected to have a longer in vivo half-life. Finally, near-IR CF dyes have much higher labeling efficiency than other near-IR dyes because of their excellent solubility and generally >95% reactivity

Advantages

Brightness: CF dyes match or surpass the brightness of Alexa Fluor, Cy and other commercially available dyes.
Photostability: CF dyes are among the most photostable dyes.
Specificity and in-vivo Stability: CF-labeled conjugates offer super specificity and improved half-life for in-vivo imaging.
pH Insensitivity: CF dyes remain highly fluorescent over a wide pH range.
Solubility: CF dyes are highly soluble in both water and a wide range of organic solvents.
Compatibility: CF dyes are spectrally similar to other commercially available dyes and compatible with common excitation sources and existing filters.
Color Selection: CF dye series offers the largest dye collection with wavelengths ranging from UV to near IR.

Advantages

In Vivo Imaging: CF reactive dyes and kits for small animal imaging
Westerns: Perform Westerns with highly cross-adsorbed antibodies to minimize cross-reactivity and background
Protein Labeling: Reactive dyes and kits for custom conjugations
Flow Cytometry: Superior non-tandem conjugates for cell staining
Microscopy: Bright and photostable antibodies or reactive dyes for multiple labeling procedures

Figure 3: Bars represent the signal-to-noise ratio of the relative fluorescence values of the geometric means from Figure 2.

Figure 4: Goat anti-mouse IgG was labeled with CF750, AlexaFluor750 or Cy7, respectively. Fluorescence values were measured on a Hitachi F-4500 fluorometer. The normalized fluorescence values are shown above.

Figure 6: J urkat cells were stained with isotype or mouse anti-human intracellular CD3 antibody followed by 1 ug of goat anti-mouse IgG conjugated with Cy5.5, AlexaFluor680, CF680 or DyLight680. Fluorescence was detected by a BD FACS Calibur in the FL4 channel. The bars represent the signal-to-noise ratio of CD3 positive fluorescence to isotype using similar degrees of labeling (DOL).

Figure 5: Goat anti-mouse IgG was labeled with CF750, AlexaFluor750 or Cy7. Absorbance of the conjugated dyes were normalized to their respective absorbance max. Cy7 and AlexaFluor750 have large shoulder peaks which are indicative of dye aggregation and do not contribute to overall fluorescence intensity. intensity.y.aaandlfandandaanodo not contribute to fluorescence.

Figure 8: Jurkat cells were stained with isotype or intracellular CD3 antibody followed by 1 ug of goat anti-mouse IgG conjugated with CF770, DyLight 800 or IRDye800CW, respectively. Fluorescence was measured on a BD LSR II equipped with a 633 nm laser and 780/60 nm PMT detector. The bars represent the relative flurorescence values of the geometric means.

Figure 7: HeLa cells were stained with mouse alpha-tubulin antibody followed by CF680 goat anti-mouse IgG. Images were captured using an Olympus mercury arc lamp microscope equipped with a Cy5 filter set, CCD camera and ImagePro Express software.

Figure 9: Two-fold dilutions of HeLa cells were cultured in a black, clear bottom 96-well plate. Cells were fixed, permeabilized and probed with mouse alpha-tubulin and rabbit COX IV primary antibodies followed by goat anti-mouse CF770 or IRDye 800 (green) and goat anti-rabbit CF680 or IRDye680 (red) at the same final concentrations. After probing, cells were dried and scanned using an Odyssey infrared imaging system (Li-Cor). The upper two rows were probed with CF dye conjugates while the lower two rows were probed with commercially purchased IRDye secondaries (Li-Cor).

Figure 10: Two-fold dilutions of HeLa cell lysate were run on an acrylamde gel, transferred to a nitrocellulose membrane and probed with mouse alpha-tubulin and rabbit COX IV primary antibodies followed by goat anti-mouse CF770 or IRDye 800 (green) and goat anti-rabbit CF680 or IRDye680 (red) at the same final concentrations. After probing, membranes were dried and scanned using an Odyssey infrared imaging system (Li-Cor Biosciences). Quantitation of the bands showed approximately a 3.5-fold higher fluorescence intensity of CF dyes compared to the respective IRDye secondary antibodies (Li-Cor).

Figure 11. Tumors in mice were imaged 24 and 96 hrs after i.v. injection of Avastin conjugated CF750 using an IVIS imaging system (Caliper LS).
Images courtesy of Caliper Life Sciences.

CF Dye	Ex/Em**	Alternative for:
CF680*	681/698	AF680,Cy5.5, DyLight680,IRDye680
CF750	755/777	AF750, Cy7
CF770*	770/797	DyLight800, IRDye800

* Compatible with the LI-COR Odyssey system ** Ex/Em wavelengths based on antibody conjugates

*CF dye technology is covered by pending US and international patents; Alexa is a registered trademark of Invitrogen.; Cy is a trademark of GE Healthcare; DyLight is a trademark of Thermo Fisher Scientific; and IRDye and Odyssey are registered trademarks of Li-COR Biosciences; in-cell Western is a trademark of Li-Cor Biosciences..

Table A. Near-IR CF Dye-labeled Secondary Antibody Conjugates

Cat#	Product Name	Unit Size	Unit Price
20292	Bovine serum albumin, CF680 conjugate	5x1mg	$190
20060	CF680 Donkey Anti-Goat IgG (H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$175
20060-1	CF680 Donkey Anti-Goat IgG (H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20241	CF680 Donkey Anti-Guinea Pig IgG (H+L), highly cross-adsorbed, 2 mg/mL	0.25 mL	$185
20241-1	CF680 Donkey Anti-Guinea Pig IgG (H+L), highly cross-adsorbed, 2 mg/mL	50 uL	$75
20278	CF680 donkey anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$180
20278-1	CF680 donkey anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20062	CF680 Donkey Anti-Sheep IgG (H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$175
20062-1	CF680 Donkey Anti-Sheep IgG (H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20287	CF680 goat anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$180
20287-1	CF680 goat anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20065	CF680 Goat Anti-Mouse IgG (H+L) (min X Human, Bovine, Horse, Rabbit, and Swine), 2 mg/mL	0.25 mL	$175
20063	CF680 Goat anti-mouse IgG (H+L), F(ab')2 fragment, 2 mg/mL	0.25 mL	$145
20065-1	CF680 Goat anti-mouse IgG (H+L), highly cross-adsorbed, 2 mg/mL	50 uL	$75
20253	CF680 Goat anti-mouse IgG1, (γ1), 2 mg/mL	0.25 mL	$210
20263	CF680 Goat anti-mouse IgG2a, (γ2a), 2mg/mL	0.25 mL	$210
20273	CF680 Goat anti-mouse IgG2b, (γ2b), 2mg/mL	0.25 mL	$210
20067	CF680 Goat Anti-Rabbit IgG (H+L) (min X Human, Mouse, and Rat), 2 mg/mL	0.25 mL	$180
20064	CF680 Goat Anti-Rabbit IgG (H+L), F(ab')2 fragment, 2 mg/mL	0.25 mL	$145
20067-1	CF680 Goat Anti-Rabbit IgG (H+L), highly cross-adsorbed, 2 mg/mL	50 uL	$75
20069	CF680 Goat Anti-Rat IgG (H+L), highly cross-adsorbed, 2 mg/mL	0.25 mL	$175
20069-1	CF680 Goat Anti-Rat IgG (H+L), highly cross-adsorbed, 2 mg/mL	50 uL	$75
20219	CF680 monoclonal mouse anti-GFP IgG, 1 mg/mL	0.1 mL	$240
20068	CF680 Rabbit Anti-Goat IgG (H+L), 2 mg/mL	0.5 mL	$165
20068-1	CF680 Rabbit Anti-Goat IgG (H+L), 2 mg/mL	50 uL	$70
20243	CF680 Rabbit Anti-Guinea Pig IgG (H+L), 2 mg/mL	0.5 mL	$175
20243-1	CF680 Rabbit Anti-Guinea Pig IgG (H+L), 2 mg/mL	50 uL	$70
20061	CF680 Rabbit Anti-Mouse IgG (H+L), highly crossed-adsorbed, 2mg/mL	0.25 mL	$175
20061-1	CF680 Rabbit Anti-Mouse IgG (H+L), highly crossed-adsorbed, 2mg/mL	50 uL	$75
20196	CF680R Donkey Anti-Goat IgG (H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$185
20196-1	CF680R Donkey Anti-Goat IgG (H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20194	CF680R Donkey Anti-Mouse IgG (H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$185
20194-1	CF680R Donkey Anti-Mouse IgG (H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20195	CF680R Donkey Anti-Rabbit IgG (H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$185
20195-1	CF680R Donkey Anti-Rabbit IgG (H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20192	CF680R Goat anti-mouse IgG (H+L), highly cross-adsorbed, 2 mg/mL	0.25 mL	$185
20192-1	CF680R Goat anti-mouse IgG (H+L), highly cross-adsorbed, 2 mg/mL	50 uL	$75
20193	CF680R Goat Anti-Rabbit IgG (H+L), highly cross-adsorbed, 2 mg/mL	0.25 mL	$185
20193-1	CF680R Goat Anti-Rabbit IgG (H+L), highly cross-adsorbed, 2 mg/mL	50 uL	$75
20298	CF750 Donkey Anti-Rabbit IgG (H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$180
20298-1	CF750 Donkey Anti-Rabbit IgG (H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20070	CF750 Goat Anti-Mouse IgG (H+L), 2 mg/mL	0.5 mL	$165
20070-1	CF750 Goat Anti-Mouse IgG (H+L), 2 mg/mL	50 uL	$70
20073	CF750 Goat Anti-Rabbit IgG (H+L), 2 mg/mL	0.5 mL	$165
20073-1	CF750 Goat Anti-Rabbit IgG (H+L), 2 mg/mL	50 uL	$70
20277	CF770 Donkey Anti-Goat IgG (H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$180
20277-1	CF770 Donkey Anti-Goat IgG (H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20242	CF770 Donkey Anti-Guinea Pig IgG (H+L), highly cross-adsorbed, 2 mg/mL	0.25 mL	$185
20242-1	CF770 Donkey Anti-Guinea Pig IgG (H+L), highly cross-adsorbed, 2 mg/mL	50 uL	$75
20279	CF770 donkey anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$180
20279-1	CF770 donkey anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20288	CF770 goat anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	0.25 mL	$180
20288-1	CF770 goat anti-human IgG(H+L), highly cross-adsorbed, 2mg/mL	50 uL	$75
20077	CF770 Goat Anti-Mouse IgG (H+L) (min X Human, Bovine, Horse, Rabbit, and Swine), 2 mg/mL	0.25 mL	$175
20077-1	CF770 Goat Anti-Mouse IgG (H+L) (min X Human, Bovine, Horse, Rabbit, and Swine), 2 mg/mL	50 uL	$75
20254	CF770 Goat anti-mouse IgG1, (γ1), 2 mg/mL	0.25 mL	$210
20264	CF770 Goat anti-mouse IgG2a, (γ2a), 2mg/mL	0.25 mL	$210
20274	CF770 Goat anti-mouse IgG2b, (γ2b), 2mg/mL	0.25 mL	$210
20078	CF770 Goat Anti-Rabbit IgG (H+L) (min X Human, Mouse, and Rat), 2 mg/mL	0.25 mL	$175
20078-1	CF770 Goat Anti-Rabbit IgG (H+L) (min X Human, Mouse, and Rat), 2 mg/mL	50 uL	$75
20220	CF770 monoclonal mouse anti-GFP IgG, 1 mg/mL	0.1 mL	$240
20244	CF770 Rabbit Anti-Guinea Pig IgG (H+L), 2 mg/mL	0.5 mL	$175
20244-1	CF770 Rabbit Anti-Guinea Pig IgG (H+L), 2 mg/mL	50 uL	$70

Table B. Other Near-IR CF dye-labeled Products

Cat#	Product Name	Unit Size	Unit Price
29007	Annexin V, CF680 conjugate, lyophilized	25 ug	$250
29006	Annexin V, CF750 conjugate, lyophilized	25 ug	$250
00048	Phalloidin, CF680R conjugate	300 U	$325
29025	Wheat Germ Agglutinin, CF680R conjugate	5x1mg	$270
29025-1	Wheat Germ Agglutinin, CF680R conjugate	1mg	$70

Table C. Reactive Near-IR CF Dyes and Labeling Kits

Cat#	Product Name	Unit Size	Unit Price
92029	CF680 maleimide	1 umole	$205
92220	CF680 SE protein labeling kit	3 labelings	$365
92139	CF680 succinimidyl ester	1 umole	$240
92032	CF680R maleimide	1 umole	$230
92226	CF680R SE protein labeling kit	3 labelings	$365
92107	CF680R succinimidyl ester	1 umole	$250
92221	CF750 SE protein labeling kit	3 labelings	$365
92142	CF750 succinimidyl ester	1 umole	$240
92222	CF770 SE protein labeling kit	3 labelings	$365
92150	CF770 succinimidyl ester	1 umole	$240
92155	CF790 succinimidyl ester	0.25 umole	$140
92282	Mix-n-Stain CF680 antibody labeling kit, 1X(5-20ug) labeling	1 labeling	$95
92283	Mix-n-Stain CF680R antibody labeling kit, 1X(5-20ug) labeling	1 labeling	$95
92241	Mix-n-Stain CF750 antibody labeling kit, 1x(50-100 ug) labeling	1 labeling	$115
92284	Mix-n-Stain CF750 antibody labeling kit, 1X(5-20ug) labeling	1 labeling	$95
92265	Mix-n-Stain CF770 antibody labeling kit, 1x(20-50 ug) labeling	1 labeling	$105
92242	Mix-n-Stain CF770 antibody labeling kit, 1x(50-100 ug) labeling	1 labeling	$115
92285	Mix-n-Stain CF770 antibody labeling kit, 1X(5-20ug) labeling	1 labeling	$95
92268	Mix-n-Stain CF790 antibody labeling kit, 1x(20-50 ug) labeling	1 labeling	$105
92248	Mix-n-Stain CF790 antibody labeling kit, 1x(50-100 ug) labeling	1 labeling	$115
92288	Mix-n-Stain CF790 antibody labeling kit, 1X(5-20ug) labeling	1 labeling	$95
92160	VivoBrite rapid antibody labeling kit for small animal in vivo imaging, CF680 SE	3 labelings	$395
92161	VivoBrite rapid antibody labeling kit for small animal in vivo imaging, CF750 SE	3 labelings	$395
92162	VivoBrite rapid antibody labeling kit for small animal in vivo imaging, CF770 SE	3 labelings	$395
92163	VivoBrite rapid antibody labeling kit for small animal in vivo imaging, CF790 SE	3 labelings	$410

**Price is for US enduser only. International price may vary. Please contact your local distributors for your price.