mFluor Dyes and Kits

mFluor™ dyes are a series of superior fluorescent labeling dyes that span the UV to near-infrared (NIR) spectrum. Developed exclusively by AAT Bioquest, mFluor™ dyes exhibit excellent aqueous solubility and large Stokes Shifts, and their hydrophilic property minimizes the use of organic solvents. mFluor™ dyes have been extensively used to label antibodies proteins and other biomolecules for multicolor flow cytometry applications. The absorbance maximas of mFluor™ dyes are designed to be optimally excited by one of the major laser lines commonly equipped in flow cytometers, such as the 350 nm, 405 nm, 488 nm, 532 nm, 561-568 nm or 633-647 nm laser lines. Compared to phycobiliproteins PE, APC and their tandems, mFluor™ dyes are excellent alternatives for immunophenotyping, FACS and other flow-based applications. mFluor™ dyes are available in a wide selection of products including reactive dyes and antibody labeling kits, as well as mFluor™ streptavidin conjugates for signal amplification and annexin V-mFluor™ conjugates for apoptosis detection.

mFluor™ Reactive Dyes

mFluor™ reactive dyes can be covalently labeled to biomolecules without self-quenching, resulting in intensely bright fluorescent conjugates. mFluor™ reactive dye formats include amine-reactive succinimidyl esters (SE) and thiol-reactive maleimides for labeling antibodies and proteins. mFluor™ dyes are also available in acid form for labeling proteins, peptides, amine-modified oligonucleotides and other amine-containing biomolecules by carbodiimide conjugation chemistry (EDAC).

Key features of mFluor™ reactive dyes

• Simple, quick and robust labeling of biomolecules with high conjugation yields
• Superior brightness, photostability and solubility
• Strong fluorescence emission over a broad pH range with little pH sensitivity
• Low spillover into most detectors
• More choices and flexibility for multicolor panel design

Figure 1. Flow cytometry analysis of HL-60 cells stained with 1 µg/mL mouse IgG control (Green) or with 1 µg/mL mouse anti-human HLA-ABC (W6/32 mAb) (Red) and then followed by goat anti-mouse IgG-mFluor™ Violet 450 conjugate. The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the Pacific Blue channel.

The table below shows the range of available mFluor™ reactive dyes, their excitation and emission maxima, and their fluorophore equivalents to assist you in selecting the best fluorophore for labeling proteins, antibodies, and other biopolymers, as well as, derivatizing low molecular weight molecules. For general antibody and protein labeling use mFluor™ SE which target primary amines (-NH₂). For site-specific cysteine-directed conjugation use mFluor™ maleimides.

ReadiLink™ Rapid mFluor™ Antibody Labeling Kits

ReadiLink™ Rapid mFluor™ Antibody Labeling Kits from AAT Bioquest provide a convenient method for labeling microscale volumes of antibodies with our superior mFluor™ dyes. The unique chemistry of ReadiLink™ kits enable researchers to effortlessly label and recover 100% of their antibodies without a purification step. Since ReadiLink™ mFluor™ conjugates are covalently labeled they are stable for long-term storage and they are ideal for demanding applications including immunophenotyping, multiplex flow cytometry, FACS and other flow-based applications. Also available are ReadiLink™ Rapid and xtra Rapid Antibody Labeling kits for conjugation of mFluor™ dyes, Alexa Fluor® dyes, other fluorescent dyes, biotin, BSA and KLH.

Key features of ReadiLink™ mFluor™ Antibody Labeling Kits

• Label monoclonal antibodies, polyclonal antibodies or other proteins
• Quick and simple two-step labeling protocol, no chemistry or conjugation experience necessary
• Useful for labeling 100 µg of antibody (two separate labeling reactions of 50 µg antibody)
• 100% antibody recovery, NO purification of the product is required
• Conjugates ready to use in 1 hr, with as little as ∼10 minutes hands-on time
• Avoid using any amine-containing buffers (e.g. Tris or glycine)
• Stabilizing proteins (e.g. BSA or gelatin) must be removed prior to labeling, for antibody labeling kits compatible with BSA use ReadiLink™ xtra Rapid Antibody Labeling Kits

mFluor™ Bioconjugates

We offer a large selection of mFluor™-labeled conjugates for a wide range of applications. Featured conjugates include, Annexin V for apoptic detection and streptavidin, which when used together with biotin can amplify weak signals in vitrually any immunoassay.

Annexin V mFluor™ Conjugates for Apoptosis Detection

Annexin V conjugated to mFluor™ dyes provide a fast and reliable method for detecting externalized PS, a key characteristic in early-stage apoptosis. The superior brightness and photostability of Annexin V mFluor™ conjugates are useful for live cell imaging, immunofluorescence, and flow cytometry, and can be combined with other dyes, such as nucleic acid stains, to accurately assess mixed populations of live, apoptotic, necortic and dead cells.

Key features of Annexin V mFluor™ Conjugates

• mFluor™ conjugates generate brighter more photostable signals
• Compatible with live cells only
• Fixable after staining with formaldehyde
• Suitable for all fluorescence-based imaging platforms
• Conjugates to match any laser setting
• Available in multicolor wavelengths for multiplexing applications

Figure 3. The detection of binding activity of Annexin V-mFluor™ Blue 570 to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 µM staurosporine (Red) at 37°C for 4 hours, and then labeled with Annexin V-mFluor™ Blue 570 conjugate for 30 minutes.

Table 3. Available Annexin V-mFluor™ conjugates for apoptosis detection.

mFluor™ Streptavidin Conjugates for Signal Amplification

Fluorescently labeled mFluor™ streptavidin conjugates have been widely used in fluorescence imaging to detect biotinylated antibodies, ligands and DNA probes for in situ hybridization techniques, immunohistochemistry, multicolor flow cytometry and other applications that require high sensitivity. mFluor™ streptavidin conjugates exhibit minimal self-quenching, producing exceptionally bright and photostable fluorescence signals outperforming Alexa Fluor® and other spectrally similar conjugates. In addition, the large Stokes shifts of mFluor™ dyes minimize cross-talk between the laser and emission filter and allows for cellular imaging with high singal-to-noise ratios. mFluor™ streptavidin conjugates are designed for optimal excitation by one of the major laser lines commonly equipped in flow cytometers, such as the 405 nm, 488 nm, 532 nm, 561 nm or 633 nm laser lines.

Figure 4. Flow cytometry analysis of HL-60 cells stained with (Red) or without (Green) 1 µg/ml Anti-Human HLA-ABC-Biotin and then followed by mFluor™ Yellow 630-streptavidin conjugate.

Table 4. Available mFluor™ streptavidin conjugates for flow cytometry.

Additional Resources

1. Alexa Fluor® is a registered trademark of ThermoFisher.
2. Horizon™ is a trademark of BD Biosciences.