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
- 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.
- 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: Jurkat 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 IRDye®800CW, 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 IRDye®680 (red) at the same final concentrations. After probing, cells were dried and scanned using an OdysseyTM 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 IRDye®680 (red) at the same final concentrations. After pro bing, membranes were dried and scanned using an OdysseyTM 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.
* 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
Table B. Other Near-IR CF dye-labeled Products
Table C. Reactive Near-IR CF Dyes and Labeling Kits
|**Price is for US enduser only. International price may vary.
Please contact your local distributors for your price.