Detection of S-phase Cell Cycle Progression using 5-ethynyl-2′-deoxyuridine Incorporation with Click Chemistry, an Alternative to using 5-bromo-2′-deoxyuridine Antibodies

Abstract

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.

Rapid proliferation of activated lymph node CD4+ T cells is achieved by greatly curtailing the duration of gap phases in cell cycle progression

“…EdU is one of the nucleoside analogs used for this purpose [13]. When EdU is added to the culture for a short enough period of time, it was possible to distinguish the CD4 + T cells that were synthesizing DNA ( Fig.…”

Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells

“…Importantly, the same limitation in interpretation applies to other methods which rely on incorporation of an analog into DNA (e.g., tritiated thymidine or fluorogenic deoxyuridine). 65 A recent study of replication marker expression in human β-cells demonstrated that co-expression of two or more markers more accurately discriminates between quiescent and replicating β-cells. 66 Expression of Ki67 is used routinely as a proliferation marker in immunohistochemical studies.…”

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

“…However, a potential alternative could be to use 5-ethynyl-2′-deoxyuridine (EdU), another thymidine analog that is, similar to BrdU, incorporated efficiently in replicating DNA 41 . Visualization of EdU using 'click chemistry" does not require acid hydrolysis 42 , and may therefore more suitable for some antibodies.…”