Thymidine stock

Stock cultures are established from frozen ampoules of cells that have been treated with thymidine, hypoxanthine, methotrexate and glycine for 24 h, which purges the culture of pre-existing TK / mutants. This cell stock is used for a maximum of two months. 

HT Stock solution Make slurry of 408 mg hypoxanthine in 100 ml ddH20, then add 1M sodium hydroxide until all solid is dissolved. Dissolve 114 mg thymidine in 100 ml ddH20. Combine both solutions, fill up to 300 ml, and adjust pH with acetic acid to 10.0. Filter through a 0.2-pm filter and store in aliquots at -20 °C. 

Reagents. Aqueous ethanol solutions of the triethylammonium salts of adenosine-5 -[a-S2P]triphosphate (a- PATP), adenosine-5 -[7-S2P]triphosphate (7-S2P-ATP), thymidine-5 -[a-S2P]triphosphate (a- p-TTP), cytidine-5 -[q-S2P]triphosphate (a-S2P-CTP), and guanosine-5 -[a-S2P]triphosphate (a-S2P-GTP) were purchased from Amersham (Arlington Heights, IL). Radioactive sample concentrations reported for detector efficiency determination were adjusted from the manufacturer s specifications after subjecting several diluted aliquots of the stock solution to liquid scintillation counting. 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

CldU, IdU, BrdU, and thymidine (Sigma, St Louis, MO cat. 19250). Prepare 0.20 mAf stock solutions of CldU, IdU, or BrdU and a 10 mM (100-fold concentrated) stock solution of thymidine. Store at 4°C. 

Label with IdU, CldU, or BrdU by adding 100 p stock solution to the 2 ml culture medium per well. Mix gently. Experience shows that minimally 2 to 3 min incubation is required to incorporate sufficient halo-dU to obtain a good fluorescence signal. After this period cells can be fixed immediately or the halo-dU label can be chased and the cells can be cultured for up to several generation times. Note that at each cell division the amount of labeled DNA per cell is reduced. To chase the halo-dU label remove the culture medium and add 1 ml prewarmed culture medium per cover slip/well, containing 100 pM thymidine. After 1 min remove the medium. Wash once with 1 ml of prewarmed medium without thymidine. Finally, add 2 ml medium and culture the cells and incubate for the desired chase period. 

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