Synthetic Thymidine Analog Labeling without Misconceptions

Abstract

Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains unresolved. Here, we address these concerns by examining the effects of BrdU and EdU on adult hippocampal neurogenesis and EdU on the perinatal somatic development of mice. We show that, in a wide range of doses, EdU and BrdU label similar numbers of cells in the dentate gyrus shortly after administration. Furthermore, whereas the administration of EdU does not affect the division and survival of neural progenitor within 48 h after injection, it does affect cell survival, as evaluated 6 weeks later. We also show that a single injection of various doses of EdU on the first postnatal day does not lead to noticeable changes in a panel of morphometric criteria within the first week; however, higher doses of EdU adversely affect the subsequent somatic maturation and brain growth of the mouse pups. Our results indicate the potential caveats in labeling the replicating DNA using thymidine analogs and suggest guidelines for applying this approach.

Neoblast-like Stem Cells ofFasciola hepatica

“…Here, juveniles were only incubated in EdU for 24 h, whereas previously juveniles were incubated in CS50 with EdU continuously for seven days [35]. Thymidine analogues have been shown in mammalian systems to disrupt the cell cycle and neurogenesis, as well as reducing cell viability [46–48].…”

Detection of De Novo Dividing Stem Cells In Situ through Double Nucleotide Analogue Labeling

“…We delivered BrdU via mini osmotic pumps at a rate of 50 mg/kg per day. This delivery rate can be considered, at most, equivalent to a single intraperitoneal injection of BrdU at a dose of 50 mg/kg per day, which does not show an apparent detrimental effect on proliferating cells [ 51 , 52 , 53 ]. Moreover, we are focused on the population of EdU + BrdU − cells (i.e., those that lack the BrdU label).…”

“…Hence, any potential detrimental effects of BrdU should not affect the cell population of our interest. Furthermore, the short chase period (2 h) after the EdU administration is unlikely to lead to potential EdU-induced cytotoxic effects, which were found to manifest primarily after one round of cell division [ 53 , 54 , 55 , 56 ]. In experiments aimed to trace the fates of de novo dividing cells, when prolonged chase periods after the second label pulse may be necessary, other thymidine analogues such as (2S)-2-deoxy-2-fluoro-5-ethynyl uridine (F-ara-EdU) can potentially be used as the second label because F-ara-EdU was shown to be less toxic than EdU or BrdU [ 57 ].…”

Perfect duet: Dual recombinases improve genetic resolution

“…Nucleotide analog incorporation (EdU or BrdU) is a conventional practice in the field. However, accompanying negative side effects including antiproliferation and cell toxicity were reported 41,42 . Additionally, proliferation markers staining is also extensively used, for instance, cell cycle marker Ki67 (Mki67), cytokinesis marker Aurora kinase B (AuroraB or AURKB), phosphorylated histone H3 (PH3) and proliferating cell nuclear antigen 43–45 .…”

“…However, accompanying negative side effects including antiproliferation and cell toxicity were reported. 41,42 Additionally, proliferation markers staining is also extensively used, for instance, cell cycle marker Ki67 (Mki67), cytokinesis marker Aurora kinase B (AuroraB or AURKB), phosphorylated histone H3 (PH3) and proliferating cell nuclear antigen . [43][44][45] Given that marker staining only reflects snapshots of cell proliferation, rather than continuous traces over a time window, still, signal interference is generated from other proliferating cells.…”