3-Carboxy-6-chloro-7-hydroxycoumarin: a highly fluorescent, water-soluble violet-excitable dye for cell analysis.
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 (approximately 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 conjugates are functional and stable.
Abrams
Barny
B
BD Biosciences, Cell Analysis, 2350 Qume Drive, San Jose, CA 95131, USA. barny_abrams@bd.com
Diwu
Zhenjun
Z
Guryev
Oleg
O
Aleshkov
Sergei
S
Hingorani
Ravi
R
Edinger
Mark
M
Lee
Rita
R
Link
Joe
J
Dubrovsky
Tim
T
D017252:CD3 Complex / Q000737:chemistry / Q000378:metabolism; D015704:CD4 Antigens / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003374:Coumarins / Q000737:chemistry*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017493:Leukocyte Common Antigens / Q000737:chemistry / Q000378:metabolism; D038761:Photobleaching; D013050:Spectrometry, Fluorescence; D014468:Umbelliferones / Q000737:chemistry*
C539571:3-carboxy-6-chloro-7-hydroxycoumarin; D017252:CD3 Complex; D015704:CD4 Antigens; D003374:Coumarins; D005456:Fluorescent Dyes; D014468:Umbelliferones; D017493:Leukocyte Common Antigens