Tritiated thymidine pulse labelling

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.

This was shown clearly by Stubblefield and Mueller (1962), who demonstrated focalised synthesis of DNA by pulse-labelling a random population of cells with tritiated thymidine and then after varied time intervals visualised autoradiographically the regions of metaphase chromosomes where incorporation has occurred. 

Detailed analysis using several different methods (Pulse and continuous label with tritiated thymidine, DNA fluorescence per cell using the fluorescent Feulgen assay and flow microfluorography (Van Dilla et al., 1969), and DNA specific fluorescence using the diaminobenzoic acid assay (Kissane and Robins, 1958) reveals that the DNA content of a cell increases in a saltatory fashion, and that the early portion of S-phase is a period of low net DNA synthesis which may be mistaken for G1 if insensitive methods of measurement are used (Klevecz et al., 1975). 

Fig. 9. Synchronization with seven heat shocks of two parallel 150-ml cultures, of which one served as a control (C). The second received methotrexate + uridine (M + U) after shock 3. After washing of both populations with fresh medium after heat shock 7, 0.05-ml aliquots were drawn from both flasks. They were pulsed for 10 minutes with tritiated thymidine, and 10 to 20=jLil samples were fixed for autoradiography. The percentage of macronuclei labeled in acid-stable compounds (DNA) were plotted at the end of the incorporation intervals (lower curves). The upper curves show the percentage of dividers against time. The control goes from 0 to 95 percent in division 1 (visual estimates). (From Zeuthen. 1970. Exp. Cell Res., 61 311 -325.)...

C3H/HeN(H-2d) cells were used to stimulate c57Bl/6N(h-2b) spleen cells, in the presence of varying doses of poly(ICLC). After 4 days of cocultivation, the mixed cultures were pulse labeled with tritiated thymidine for 24 hours and the incorporated radioactivity determined. All doses from 0.01 pg/mL to 5 pg/mL of poly(ICLC) stimulated the MLR, but 10 pg/mL caused inhibition.23... 

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