Thymidine autoradiography

Gaviness, V.S., Jr. (1982) Neocortical histogenesis in normal and reeler mice a developmental study based upon PH] thymidine autoradiography. Dev Brain Res 4 293-302. 

Shimada, M. (1966) Cytokinetics and histogenesis of early postnatal mouse brain as studied by H thymidine autoradiography. Arch. Histol. Japon., 4, 413-437. 

Favoli M A, Grilli CM. Autoradiography and ultrastructural study of Nostoc species labeled with tritialed thymidine. Microbios 1986 45 33-40. 

The use of autoradiographic methods confirmed bidirectional replication. Strains of amino acid aux-otrophs with small nucleoside triphosphate pools were used. The addition of amino acids after starvation led to initiation of replication with only a 6-min lag. The cells were labeled with [3H]thymidine, and after the replication forks had moved a short distance from the origin of replication the cells were given a pulse of "super-hot" [3H]thymidine. Using autoradiography it was possible to observe the clearly bidirectional replication forks378 (Fig. 27-17). Replication in other bacteria is also bidirectional. 

Cell kinetics is defined as the measurement of time parameters m biological systems. Traditionally, this has involved the use of radioactive precursors of DNA, such as tritiated thymidine (3HTdR), and autoradiography to detect their incorporation into DNA. This technique has provided detailed knowledge of cell kinetics in both in vitro and in vivo experimental systems. The technique, however, is time consuming and arduous and is not readily applicable to human tumor research because of ethical problems involved in incorporation of a radioisotope into DNA. 

Autoradiography of contaminated cultures labelled with tritiated thymidine shows grains over the whole cell rather than localised to the nucleus. This is a result of degradation of the thymidine to thymine followed by incorporation into mycoplasma DNA and other cell constituents. 

Fig. 10.2. Mitotic stages. Mouse L929 cells, pulse labelled with tritiated thymidine, were processed for autoradiography. Among the S-phase cells (covered with grains) are cells in other stages of interphase and several mitotic cells. A metaphase cell, two anaphase cells and a late telophase cell are distinguishable.

Fig. 10.13. Distribution of cells separated by flow microfluorometry. CHO cells were pulse labelled for 15 min with [3H]thymidine (1 / Ci/ml) and stained with ethidium bromide. They were then submitted to flow microfluorometry and cell sorting on the basis of cellular DNA content. Cells from the indicated portions (sort 1, 2 and 3) were then subjected to autoradiography and shown to contain respectively 4%, 93% and 19% of the cells labelled. (Reproduced from Kraemer et al., 1973, with kind permission of the authors and publisher.)...

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

Fig. 12.6. Grain count distribution. L cells labelled for 10 min with [3H]thymidine (2.5/1 Ci/ml 0.36 Ci/mmol) and processed for autoradiography using NTB3 emulsion. Those cells (62% of the total) with one or more grains are recorded. A Poisson distribution with a mean of 30 is included for comparison. (Reproduced from Cleaver, 1967, with kind permission of the author.)...

The semiconservative replication of DNA at the chromosomal level was shown by J. H. Taylor and coworkers. Using autoradiography and bean seedling root cells in tissue culture, they showed that, after a part of a cycle of duplication with [3H]thymidine (a selective label for DNA), the two chromosomes, descended from an original unlabeled chromosome, were both labeled. Following an additional duplication in the absence of labeled thymidine, the labeled chromosome yielded one labeled and one unlabeled descendant, as predicted by the semiconservative mechanism. 

Liver cells are then exposed in vitro to the test compound and incubated with tritium-labeled thymidine for about 18 hours. At the end of the incubation, the cells are fixed on slides and prepared for autoradiography. For that the slides are first exposed to liquid photographic emulsion, air-dried and following a 7-day exposure in the dark, exposed to developing solution. 

Autoradiography is a technique for locating radioactive compounds within cells it can be conducted with light or electron microscopy. Living cells are first exposed to the radioactive precursor of some intracellular component. The labeled precursor is a compound with one or more hydrogen ( H) atoms replaced by the radioisotope tritium (3H) e.g., [3H]thymidine is a labeled precursor of DNA, and [3H]uridine is a labeled precursor of RNA (Chap. 7). Various tritiated amino acids are also available. The labeled precursors enter the cells and are incorporated into the appropriate macromolecules. The cells are then fixed, and the samples are embedded in a resin or wax and then sectioned into thin slices. 

Assays that detect the ability of chemical carcinogens or UV or ionizing radiations to induce unscheduled DNA synthesis, or DNA repair, are also commonly used. In one of these, autoradiography is used to measure the incorporation of tritiated thymidine into the DNA of cells treated with these carcinogens. All the assays mentioned above are commonly used at the same time to detect the geno-toxic properties of chemicals or radiation in what is referred to as a battery of tests. This battery of tests usually constitutes what is termed a primary screen... 

The first detailed study of normal human epidermal population and cellular kinetics using tritiated thymidine and autoradiography was reported by Epstein and Maibach in 1965 (E5). A detailed discussion of this study is in order since many of the concepts involved in such studies are well illustrated. Reference to Fig. 3 will make the discussion easier to follow. 

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