From "Click" to "Fenton" chemistry for 5-bromo-2'-deoxyuridine determination.
Ascorbic acid (AA) and copper have been increasingly employed in flow cytometry (FCM) and high content analysis (HCA) since the introduction of "click chemistry" as a non-destructive alternative to classical 5-bromo-2'-deoxyuridine (BrdU) immunodetection for DNA synthesis and proliferation assays. Mixtures of ascorbate and catalytic copper, under certain experimental conditions, act as oxidizing agent, catalyzing the formation of reactive hydroxyl radicals through hydrogen peroxides decomposition via Fenton reaction. We developed a procedure for BrdU incorporation detection based on the use of AA and cupric ions as DNA damaging agents. Optimal DNA damaging conditions were identified and found to provide results comparable with "click" 5-ethynyl-deoxyuridine (EdU) cycloaddition approach and classical BrdU immunodetection. Scavenger agents were found to prevent hydroxyl-induced DNA damages, providing the proof-of-concept for the use of this procedure for DNA denaturation prior to BrdU detection. We demonstrated hydroxyl radicals' reaction to be readily applicable to HCA and FCM assays, for both classical BrdU immunostaining and EdU cycloaddition procedure. This technique was successfully employed for BrdU pulse-chase experiments and in multiparametric immunofluorescence assays for the simultaneous detection of labile phosphoproteins in intact cells. The use of AA/Cu prior to immunodetection for BrdU incorporation assays is a viable alternative to chemical/physical DNA denaturing agents (acids or heat), since it allows preservation of labile epitopes such as phosphoproteins, and over enzymatic agents (digestion with DNases) for its lower cost.
Department of Biology Nerviano Medical Sciences, Via Pasteur 10, 20014, Nerviano, Italy.
Paolo
P
Cappella
Vincenzo
V
Giansanti
Maurizio
M
Pulici
Fabio
F
Gasparri
D001205:Ascorbic Acid / Q000737:chemistry*; D001973:Bromodeoxyuridine / Q000737:chemistry*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D057930:Click Chemistry; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D004261:DNA Replication / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D013194:Staining and Labeling
D004247:DNA; D001973:Bromodeoxyuridine; D001205:Ascorbic Acid
BrdU; Fenton chemistry; ascorbic acid; click chemistry; high content analysis