A fluorophore is a fluorescent chemical that can re-emit light upon excitation. Fluorophores can be used in conjugation with protein or oligonucleotides to generate fluorescent markers for detecting the expression of proteins and nucleic acid. Fluorescent labels functionally accept light energy of a given wavelength (e.g., from a laser) and re-emit energy at longer wavelength. These two processes are known as excitation and emission.
Different types of fluorophores are used in flow cytometry based on the instrument setting (see lasers and detection channels in Figure 1), the feature of fluorophores, and markers needed in designated panels such as four, six, or ten color panels.
Dyes such as FITC, PE, APC and PerCP have become the standard for many years. Tandem dyes, comprising a small fluorophore covalently coupled to a larger fluorophore, provide maximum utilization of specific equipment, increased detection channels for each laser, and generate more information for valuable, sometimes very limited, samples.
Commonly used fluorophores for surface or intracellular epitope detection are described in Figure 2.
There are many fluorophores that have potential applications in flow cytometry. A new generation of small molecule fluorophores such as Alexa Fluor, iFluor, and mFluor are rapidly replacing many traditional fluorophores, providing users with greater light stability and brighter fluorescence. CBI offers these high-performance fluorescent dyes: iFluor™ and mFluor™ series.
iFluor™ fluorophores are optimized for strong fluorescence, high photostability and pH independence, and are available to span the full UV-visible-IR spectrum. iFluor™ 488 has spectral properties essentially identical to Alexa Fluor® 488. iFluor™ 488 conjugates are prepared using a highly purified single rhodamine isomer which significantly improves lot to lot consistency. iFluor™ 647 dyes are spectrally similar to Alexa Fluor® 647 and DyLight™ 650 dyes.
mFluor™ fluorophores are developed specifically for flow cytometry applications, to enable optimal multicolor detection. These dyes show large Stokes shifts and excellent water solubility. mFluor™ dyes span the full UV-visible spectrum and are designed to maximize excitation efficiency with one of the major light sources in flow cytometers such as the violet laser (405 nm) or the blue laser (488 nm). They are excellent alternatives to the phycoprotein-based tandem dyes that are quite difficult to couple to antibodies or other biomolecules.
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Caprico manufactures RUO and ASR flow cytometry reagents. With strong expertise on bioconjugation, scientists at Caprico also provide antibody conjugation services of more than 20 different type of fluorophores, including FITC, R-PE, PerCP, APC, PerCP-Cy5.5, PE-Cy5, PE-Cy7, APC-Cy7, Biotin, mFluor450, mFluor540, iFluor488, iFluor594, iFluor647, iFluor680, iFluor700, iFluor790, PE-iFluor594, APC-iFluor700.