3-Carboxy-6-chloro-7-hydroxycoumarin: A highly fluorescent, water-soluble violet-excitable dye for cell analysis Barny Abrams a, * , Zhenjun Diwu b , Oleg Guryev a , Sergei Aleshkov c , Ravi Hingorani c , Mark Edinger a , Rita Lee a , Joe Link a , Tim Dubrovsky a a BD Biosciences, Cell Analysis, 2350 Qume Drive, San Jose, CA 95131, USA b ABDBioquest, Sunnyvale, CA 94085, USA c BD Biosciences, Cell Analysis, San Diego, CA 92121, USA article info Article history: Received 5 November 2008 Available online 24 December 2008 Keywords: Cell analysis Immunophenotyping Violet-excitable dye Multicolor flow cytometry Lymphocytes Subsetting abstract In our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays. Additional studies were performed, with BD Horizon V450–antibody conjugates being included in eight-color cocktails aimed at subsetting lymphocytes and myeloid cells. Such cocktails can frequently be unstable due to the tendency of one or more components to lose structural integrity, photobleach, or develop unwanted affinities for another component. However, the cocktails employed in this study enabled several different applications to be run and established that multicolor reagent mixtures containing V450–antibody con- jugates are functional and stable. Ó 2009 Elsevier Inc. All rights reserved. Coumarin is a naturally occurring benzopyran found in plants. Many of its derivatives are highly fluorescent. The 7-amino-4- methyl-coumarin derivatives were widely studied during the 1970s and 1980s and were found to be useful for labeling biologi- cal molecules. For example, the three-acetic acid derivative known as AMCA (7-amino-4-methylcoumarin-3-acetic acid) 1 (Fig. 1), which has a carboxyl group available for activation and an excitation maximum at 350 nm, became a popular ultraviolet (UV)-excitable fluorescent probe for labeling proteins [1]. AMCA and its more recent descendant, Alexa Fluor 350, ex- hibit an intense blue fluorescence with a narrow emission peak in the range of 440–460 nm and have exceptional photostabili- ty; compared with fluorescein, AMCA is able to tolerate illumi- nation more than three times longer. Moreover, the fluorescence intensity of AMCA is not affected by changes in pH over the range of 3–10. This is in marked contrast to other organic dyes, such as fluorescein isothiocyanate (FITC), that dis- play considerable variability in their fluorescence intensities with pH [2–4]. Although the emission peak of AMCA does not overlap with the emission peaks of other major fluorescent probes and its quantum yield (QY) is not unusually low, its excitation maximum falls in an area of high cellular autofluorescence, sometimes making it appear to be dim relative to the background. This is due to the nature of cellular autofluorescence, which is largely the result of excitation of naturally occurring components such as flavins and nicotin- amide adenine dinucleotide (reduced form) (NADH). These com- pounds are excited most efficiently in the UV region, so the laser used to excite AMCA also produces significant fluorescence from unstained cells. The emission characteristics of this autofluores- cence are broad throughout the visible spectrum (e.g., blue, green, yellow, red) such that virtually none of the common dichroic mir- ror and bandpass filter combinations used in flow cytometry detec- tion is able to eliminate it. The impact of this broad autofluorescence on flow cytometry becomes apparent with a den- sely expressed marker such as CD8 on mouse T-cells, when the sig- nal-to-noise ratio of CD8 AMCA is significantly lower than that of CD8 FITC, because the UV laser used to excite AMCA produces much higher autofluorescence than the blue laser used to excite FITC [5]. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.12.018 * Corresponding author. Fax: +1 408 954 2156. E-mail address: barny_abrams@bd.com (B. Abrams). 1 Abbreviations used: AMCA, 7-amino-4-methylcoumarin-3-acetic acid; UV, ultra- violet; FITC, fluorescein isothiocyanate; QY, quantum yield; NADH, nicotinamide adenine dinucleotide (reduced form); DMF, dimethylformamide; mAb, monoclonal antibody; MWCO, molecular weight cutoff; NHS, N-hydroxysuccinimide; D/P, dye-to- protein ratio; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FSC, forward light scatter; SSC, 90° orthogonal light scatter; PMT, photomultiplier tube; HPLC, high-performance liquid chromatography; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; APC, allophycocyanin. Analytical Biochemistry 386 (2009) 262–269 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio