Detection of bromodeoxyuridine incorporation by alteration of the fluorescence emission from nucleic acid binding dyes using only an argon ion laser.
A method was developed that uses paired DNA dyes to detect the incorporation of bromodeoxyuridine (BrdUrd) into cellular DNA and requires only 488 nm excitation. The fluorescence of thiazole blue, TO-PRO-3, and LDS-751 was found to be enhanced by the presence of BrdUrd in DNA. Pairing LDS-751, thiazole blue, or TO-PRO-3 with propidium iodide (PI) for flow cytometry allowed the differentiation of cells containing BrdUrd from BrdUrd unlabeled cells. LDS-751 can be excited directly at 488 nm, and TO-PRO-3 or thiazole blue can be excited indirectly by resonance energy transfer from PI. The enhancement of fluorescence from these dyes is correlated with a decrease in PI fluorescence, suggesting an increased energy transfer from PI to the red emitting dye. Fluorescence from other dyes, including thiazole orange, TO-PRO-1, rhodamine 800, oxazine 750, and 7-aminoactinomycin D, was not altered by the presence of BrdUrd in DNA. Results also were obtained showing that BrdUrd detection using PI and TO-PRO-3 is compatible with immunofluorescence staining with FITC-labeled antibodies.
Frey
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Becton Dickinson Immunocytometry Systems, San Jose, California 95131.
D001973:Bromodeoxyuridine / Q000032:analysis*; D002460:Cell Line; D002469:Cell Separation; D004247:DNA / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D007834:Lasers; D011419:Propidium
D005456:Fluorescent Dyes; D011419:Propidium; D004247:DNA; D001973:Bromodeoxyuridine