5‐Ethynyl‐2′‐deoxyuridine labeling detects proliferating cells in the regenerating avian cochlea

Abstract

Objectives/Hypothesis-The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of BrdU, a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA Synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using EdU as a thymidine analog and a non-antibody azide/alkyne reaction between the EdU and the fluorescent probe. We propose that EdU is as effective as BrdU but without the requirement for harsh denaturation or the use of antibodies for detection.Study Design-Two week-old chicks received a single gentamicin injection followed by a single EdU injection 72h later. Cochleae were extracted 4-8h later, fixed, and processed for fluorescent detection of EdU.Methods-Cochleae were processed for detection of incorporated EdU using the Click-iT™ Imaging Kit (Invitrogen) and co-labeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy.Results-Supporting cells incorporated EdU into their newly synthesized DNA during the 4-8h following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions-TheEdU method is as effective as BrdU without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the non-antibody EdU system allows more flexibility by enabling co-labeling with multiple antibodies to other cellular proteins involved in regeneration.

Non-destructive characterisation of mesenchymal stem cell differentiation using LC-MS-based metabolite footprinting

“…For instance, deoxyuridine is involved in the synthesis of nucleosides, the structural subunits of nucleic acids, 37 and are known to fluctuate during DNA synthesis or the S-phase of the cell cycle. 38,39 Similarly, orotidine is involved in nucleotide synthesis through pyrimidine synthesis (KEGG pathway; MAP00240). In our study, LC-MS-based footprinting detected changes in deoxyuridine and orotidine in culture medium conditioned by MSCs.…”

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

“…The mild labeling of EdU in mtDNA allows for direct comparisons with additional cellular markers [9][10][11] and enhances the utility of this technique over other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), which requires a harsh treatment to recover its epitope within DNA. Our laboratory [11][12] and others [13][14][15] have successfully used BrdU to label mtDNA.…”

“…EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU [5][6][7] does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers [9][10] . The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases.…”

Detection of S-Phase of Cell Division Cycle in Plant Cells and Tissues by Using 5-Ethynyl-2′-Deoxyuridine (EdU)

“…In addition, the mild non-caustic EdU proliferation assay components keep the proteins intact, allowing their parallel immunocytochemical detection with fluorescence-labeled antibodies. Although initially developed for application in cultured mammalian cells, the EdU assay has been successfully applied in a wide variety of species including bacteria [17], yeast [18,19], and a broad spectrum of animals [20][21][22][23] and plants [16]. The EdU-based assay has already been applied in several plant tissues and organs such as Arabidopsis root tips [24][25][26][27][28][29], leaf/petiole junction [30], and inflorescence [31].…”