Circulation time and dynamics of 3H-thymidine incorporation into DNA at various stages of chick embryo development

The most adequate method for carrying out radioautographic investigation on proliferation in the chorioallantoic tissues and in the chick embryo proper is dropping 3H-thymidine on the membrane under the shell in the area where the chorioallantois grows under. Using the application method, the radioautographic analysis of proliferation could be performed by the saturation method.     

DNA synthesis by cultured lymphocytes: a modified method for measuring 3H-thymidine incorporation

A rapid, convenient and inexpensive method for harvesting lymphocyte cultures and measuring the incorporation of ³H-thymidine into trichloroacetic acid precipitable material has been developed. The basic principle is to adsorb the entire contents of a microculture well onto the cotton applicator portion of a Q-tip, precipitate the DNA, wash away unincorporated ³H-thymidine, and count the remaining ³H in a mixture of scintillation fluid plus detergent. Data presented for mixed lymphocyte cultures between allogeneic rat lymph node cells, mixed lymphocyte cultures of human peripheral blood lymphocytes, Con A stimulated mouse spleen cells, and PHA stimulated mouse spleen cells show the method to be highly reproducible with standard deviations of less than 15% of the mean for quadruplicate mixed lymphocyte cultures and in most cases less than 5% of the mean for duplicate mitogen stimulated cultures. This culture system also gives positive values for PHA stimulated DNA synthetic responses of mouse spleen cells cultured in RPMI-1640 plus penicillin and streptomycin but without exogenous serum.     

Inhibition of 3H-thymidine incorporation by hydroxyurea: atpical response of mycoplasma-infected cells in culture.

Contamination with Mycoplasma hyorhinis was demonstrated in long-term cultures of HeLa, BICR/M1R_K rat mammary tumor, and NV1C rat neurinoma cells, by microbiological, equilibrium sedimentation, and autoradiographic techniques. In non-infected DNA-synthesizing cells, hydroxyurea (HU) in concentrations ? 10^?4 M typically inhibits ³H-thymidine (³H-TdR) incorporation into acid-insoluble material. This effect was lacking in the contaminated cell lines, although HU did block nuclear DNA replication, as shown by pulse-cytophotometric analyses. The response to HU could be restored to normal by supplementing the culture medium either with the anti-mycoplasma agent Tylosin or with fresh rat serum. The total ³H-activity in non-infected (or anti-mycoplasma treated) versus infected cells, in the absence of HU, was up to four times higher in the former. The data indicate that (i) incorporation of ³H-TdR into the nuclear DNA of contaminated cells was strongly reduced, probably due to a ‘scavenger effect’ (i.e. utilisation and rapid cleavage) by the mycoplasma; (ii) mycoplasmal ³H-TdR incorporation, contrary to nuclear DNA replication, was insensitive to HU in concentrations ? 10^?2 M. If equally valid for other species of mycoplasma, the observed phenomenon provides a criterion (together with the possibility of a rapid test) for the presence of mycoplasmal contamination in cell cultures.     

Transferrin and iron in cultured chick embryonic neurons: a comparison between human and chick transferrins

Transferrin was not required for the short-term survival of cultured chick retinal neurons. Both human and chick transferrin failed to enhance the in vitro survival of 8- or 11-day embryonic chick retinal neurons when cultured in a defined medium. Furthermore, maintenance of neurons in the presence of chick transferrin antibody did not alter in vitro survival. Retinal neurons, however, could bind and internalize human or chick transferrin when assayed for by fluorescence immunohistochemical techniques. Binding and internalization of chick transferrin appeared to be greater than human transferrin. Iron uptake was measured in cultures maintained in the absence of transferrin. After incubation with 59FeCl3, iron uptake was 3.5 +/- 1.1 fmoles/cell. The presence of chick transferrin antibody did not significantly alter the amount of iron uptake occurring in this assay. In a comparison of human and chick transferrin mediated iron uptake, chick transferrin was 50% more effective than human transferrin in transporting iron. This study demonstrates that cultured embryonic retinal neurons are not dependent on transferrin for survival or iron uptake, although they actively bind and internalize transferrin. Results also demonstrate that whereas cultured chick retinal neurons can bind and utilize human transferrin, they do so with less efficiency than chick transferrin.     

In vitro determination of myeloma cell resistance to melphalan using the 3H-thymidine incorporation technique.

Plasma cells derived from marrow aspirates of 21 untreated myeloma patients have been cultured in RPMI 1640 medium containing 3H-thymidine and melphalan. Thereafter plasma cell-labelling index was determined autoradiographically. In 20 patients melphalan caused a measurable decrease in the percentage of labelled plasma cells. Decreases in labelling index ranging from -4% to 3% have been considered as indicating the resistance of myeloma cells to melphalan in vitro. Decreases within this range have been found in 7 patients. In other 13 myelomas with chemosensitive marrow plasma cells, melphalan did reduce the labelling index to values ranging from 4% to 24%. Some clinical implications of the tests are discussed.     

Simultaneous localization of 3H-thymidine incorporation and acid phosphatase activity in mouse spleen: EM radioautography and cytochemistry.

Simultaneous localization of 3H-thymidine incorporation and acid phosphatase (AcP) activity was undertaken by combined radioautography and cytochemistry in the spleen of mice at different ages. The localization of radiolabelled thymidine was used to determine the site of DNA synthesis (cell proliferation), while AcP activity as a marker for cell lysis/death. For EM radioautography (EMRAG), the tissue sections were incubated in a medium containing 3H-thymidine and processed for radioautography, while the lanthanide-based method for the ultrastructural localization of AcP activity was employed. Quantitation of AcP activity was carried out by X-ray microanalysis. In all tissue sections examined, mostly of the labelled nuclei were observed in the hematopoietic cells. Few mitochondria of these cells were labelled. The labeling index was expressed as the percentage of labelled cells over the total number of counted cells. The labeling indices dropped considerably from day one after birth and progressively until the 10th month. The result of AcP activity correlated well with the result of a previous work (Olea, 1991). The localization of radiolabelled thymidine and AcP activity were not hindered by the simultaneous exposure of the same tissue section to 3H-thymidine and AcP cytochemical media. Interestingly enough, the spleen actively participates both in hematopoiesis and erythrophagocytosis. Prominently, it is most active during the early postnatal life. However, their influence declined considerably at the later stage of life (adult stage).     

3H-thymidine incorporation by interstitial myocardial cells in the rat with a single administration of the label

The hearts were taken 1, 2, 3, 4, 5, 6, 7, 14 and 21 days after a single injection of 3H-thymidine into 20 young male Wistar rats. Label incorporation was determined on half-thin slices derived through the whole left ventricular wall. First labeled nuclei are seen on the day 1 at the level of 0.4 +/- 0.08%, their quantity reaches maximum (1.66 +/- 0.1%) on day 4 and declines to 0.51 +/- 0.1% at day 7. On day 21 0.1 +/- 0.14% cells contained radioactivity. Maximal label concentration was found in middle intramural layer (2.44 +/- 0.24% on day 4), and reached only 1.36 +/- 0.36% in subendocardial and 1.16 +/- 0.24% in subepicardial layer. These data allow us to suppose the existence of linkage between interstitial cells repopulation and functional activity of different myocardial layers, and also the existence of short living and long living fibroblast subpopulations.     

Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum)

The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations.     

Dynamics of 3H-thymidine and 3H-deoxycytidine incorporation into the nuclei of a rabbit kidney cell culture during the S period

Using a combined cytophotometric-autoradiographic method a study was made of 3H-thymidine and 3H-deoxycytidine incorporation rates into the interphase nuclei of rabbit kidney cell culture during the S-period. The rate of 3H-deoxycytidine (10(-4) M--10(-6) M) incorporation into nuclei increases throughout the first part of the S-period and decreases from its middle to the end. The patterns of variations of 3H-thymidine and 3H-deoxycytidine incorporation rates into the nuclei of cultured rabbit kidney cells during the S-period were identical.     

The morphological and functional characteristics of human aortic endothelium. II. Cellular polymorphism and the incorporation of 3H-thymidine in a culture]

The content of multinuclear endothelial cells and the ability of cells to incorporate 3H-thymidine were studied in primary cultures isolated from zones of low (LP) and high (HP) probability of atherosclerosis of adult human aortas. It was found that the percentage of multinuclear EC was at mean 2-fold higher in cultures from HP zones compared to LP zones of the same vessels. In primary cultures and in the first passage cultures only small mononuclear EC were able to incorporate 3H-thymidine. A significant decrease in the thymidine index (TI) was found only in cultures from HP zones of atherosclerotic aortas. In cultures of EC from the LP zones of these aortas the TI was as high as in cultures from the LP and HP zones from grossly normal vessels.     

The subcellular fractionation of embryonic chick tendon and cartilage cells: a re-examination.

A re-examination of the subcellular fractions obtained from matrix-free chick tendon and cartilage cells has been made since the discovery that three out of four of the micrographs of chick tendon microsomal fractions published in an earlier paper from this laboratory were not authentic. The present studies demonstrate that by using the procedures previously reported it is possible to isolate microsomal and submicrosomal fractions from tendon and cartilage cells which exhibit typical morphology when examined by electron microscopy. These observations are consistent with our original biochemical characterization of subcellular fractions, which we know to be valid. Other publications from this laboratory in which these fractionation procedures have been applied to studies of collagen biosynthesis are in no way compromised, and indeed, most of our data have been confirmed by several other laboratories.     

Effects of calcitonin and calcitonin gene-related peptide on 3H-thymidine incorporation into DNA of rat thyroid lobes in vitro.

The effects of a 4 h incubation of rat thyroid lobes, in the presence of calcitonin (CT) and calcitonin gene-related peptide (CGRP) on the incorporation of 3H-thymidine into DNA, were investigated. In other groups the thyroid lobes were incubated during exposure to CT and thyrotropin (TSH), and to CGRP together with TSH. All concentrations of CT (10(-6)-10(-8) M) revealed a tendency towards lowering 3H-thymidine uptake, but the effect was not statistically significant. The influence of CGRP was dose-dependent; the lowest concentration of CGRP (10(-9) M) significantly enhanced DNA synthesis in the incubated rat thyroids; an intermediate dose of the peptide (10(-8) M) had no effect, while the highest concentration of CGRP (10(-7) M) decreased 3H-thymidine incorporation. Calcitonin (10(-7) M), as well as CGRP (10(-8) M), suppressed the stimulatory effect of TSH on 3H-thymidine incorporation.     

Differential incorporation of 3H-thymidine into DNA in cultured skin fibroblasts derived from patients with cystic fibrosis and controls.

Tritiated thymidine (³H-TdR) incorporation into DNA of the fibroblasts derived from subjects with cystic fibrosis (CF) and their controls was studied with scintillation counting and autoradiography. ³H-TdR incorporation at 24 hours postseeding was significantly less (p<0.005) in CF strains in comparison with cells from controls. The percentage of labeled fibroblasts was not significantly different between the two strains (p>0.1). The cell cycle time and the duration of each phase were studied by a mitotic selection and scintillation counting technique. There was no difference in cell cycle time between CF and control fibroblasts, however, the duration of the synthetic phase was significantly (p<0.005) longer in CF subjects.