Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview

Synthetic Thymidine Analog Labeling without Misconceptions

“…Overall, our results indicate that low doses of EdU and BrdU (up to 20–25 mg/kg) are sufficient for labeling the neural progenitor cells. This stands in contrast to some of the previous findings, which indicate a pronounced dependence of the number of labeled cells in the adult neurogenic zones on the doses of administered analogs; for instance, several reports have indicated that doses of 150–200 mg/kg of BrdU are saturated for the purpose of following adult hippocampal neurogenesis [ 26 , 27 , 28 , 29 ]—for review, see [ 1 , 2 ]. The differences in the studies’ findings might potentially be due to the improved methods of detection and their specificity and sensitivity, increased sensitivity of the detection protocols, affinity of antibodies, quantum yield of the fluorescent label, and other methodological details.…”

“…Marking proliferating cells by tagging their duplicating DNA with synthetic thymidine analogs has become a key approach in cell and developmental biology and neurobiology [ 1 , 2 , 3 , 4 ]. Although other widely applied markers of dividing cells, such as Ki67, PCNA, Mcm2, or phosphorylated histone H3, are highly useful, their precision and sensitivity are lower than those of the nucleotide-based analysis; moreover, unlike nucleotide tags, they cannot be used to trace the progeny of labeled cells [ 5 ].…”

“…Although other widely applied markers of dividing cells, such as Ki67, PCNA, Mcm2, or phosphorylated histone H3, are highly useful, their precision and sensitivity are lower than those of the nucleotide-based analysis; moreover, unlike nucleotide tags, they cannot be used to trace the progeny of labeled cells [ 5 ]. The halogenated thymidine analog 5-bromo-2′-deoxyuridine (BrdU) is the most widely used analog for labeling duplicating DNA [ 1 , 2 , 3 ]. However, detecting incorporated BrdU-labeled residues with antibodies requires a lengthy procedure that is not always easily standardized for different cell and tissue types.…”

“…However, detecting incorporated BrdU-labeled residues with antibodies requires a lengthy procedure that is not always easily standardized for different cell and tissue types. In addition, in many cases, being able to mark select stages of the division cycle or trace cells’ progeny by using two or more DNA labels is highly advantageous; however, most of anti-BrdU antibodies cross-react with other’s halogenated nucleotides, such as 5-iodo-2′-deoxyuridine and 5-chloro-2′-deoxyuridine [ 2 , 6 , 7 , 8 , 9 ], thus complicating the interpretation of the results.…”

“…The terminal alkyne-bearing nucleotide 5-ethynyl-2′-deoxyuridine (EdU) has gradually become the analog of choice for both single- and multilabel experiments [ 2 , 4 , 7 , 8 ]. EdU residues incorporated in DNA can be readily identified through their click chemistry reaction with fluorescent azides, which are small-molecule compounds (unlike anti-BrdU antibodies) and, therefore, rapidly and efficiently penetrate tissue sections and entire organs [ 4 , 10 ].…”