Methods for Inferring Cell Cycle Parameters Using Thymidine Analogues

SIMPLE SUMMARY: Several procedures have been developed to infer cell-cycle time and the duration of the cycle phases, including tritiated thymidine autoradiography and the immunodetection of thymidine analogues after their incorporation into replicating DNA. These methods have supplied important insights to reveal, on fixed tissue sections and using light or electron microscopy, the proliferative behavior of different cell types under different experimental contexts. Thymidine analogous labeling has provided knowledge about cell growth kinetics that would never have been obtained by histological methods alone. ABSTRACT: Tritiated thymidine autoradiography, 5-bromo-2′-deoxyuridine (BrdU) 5-chloro-2′-deoxyuridine (CldU), 5-iodo-2′-deoxyuridine (IdU), and 5-ethynyl-2′-deoxyiridine (EdU) labeling have been used for identifying the fraction of cells undergoing the S-phase of the cell cycle and to follow the fate of these cells during the embryonic, perinatal, and adult life in several species of vertebrate. In this current review, I will discuss the dosage and times of exposition to the aforementioned thymidine analogues to label most of the cells undergoing the S-phase of the cell cycle. I will also show how to infer, in an asynchronous cell population, the duration of the G(1), S, and G(2) phases, as well as the growth fraction and the span of the whole cell cycle on the base of some labeling schemes involving a single administration, continuous nucleotide analogue delivery, and double labeling with two thymidine analogues. In this context, the choice of the optimal dose of BrdU, CldU, IdU, and EdU to label S-phase cells is a pivotal aspect to produce neither cytotoxic effects nor alter cell cycle progression. I hope that the information presented in this review can be of use as a reference for researchers involved in the genesis of tissues and organs.