Streptomycin stock

Saos-2 cell culture growth medium. 420 mL McCoy s 5 A (ATCC) supplemented with 1.5 mM glutamine, 1.1 g sodium bicarbonate (final concentration 2.2 g/L), 5 mL penicillin and streptomycin stock solution (ree Subheading 2.1) (100 times diluted to have final 100 U/mL penicillin and 0.1 mg/mL streptomycin) and 75 mL fetal bovine serum from PAA see Subheading 2.1), final concentration of bovine serum is 15 % v v. 

Cell Culture. KB cells were maintained in a humidified atmosphere of 5% carbon dioxide - 95% air at 37°C in the presence of modified Eagle s medium containing calf serum (10%), penicillin (100 jug/ml) and streptomycin (100 units/ml). Cells were routinely subcultured with 0.25% trypsin and stocks were discarded after twenty passages. All drugs were administered with fresh media 24 hours after subculture in the following concentrations TPA, 1.6 uM RA, 1.6 juM butyric acid, 2mM. Drug treatments were for 20-24 hours. Cells were harvested for enzyme assays with phosphate-buffered saline containing 0.05% EDTA and stored at -20°C in 0.32 M sucrose. 

Dulbecco s modified Eagle s medium (DMEM) with L-glutamine and 4.5 gm/L glucose. Add penicillin-streptomycin (Gibco BRL, Gaithersburg, MD) from 100X stock. Add fetal bovine serum (FBS) to a final concentration of 10%. 

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. 

Glycerol stock cultures should be kept at -80 °C. Bacteria for experiments should be picked from colonies grown on LB plates not more than 1 week old and stored at 4 °C. The SL1344 strain is streptomycin resistant, so can be grown on LB plates containing 100 pg/mL streptomycin. 

T3 Growth Media (store at 4°C) The following are added to DMEM (formulation containing 1000 mg/L glucose and 2 mM L-glutamine) to give the final indicated concentrations 10% FBS 100 U/mL penicillin 1000 pg/mL streptomycin. 8 mg/mL Polybrene stock solution in PBS. 

Fig. IB. The effect of SAB on the repair kinetics of potentially lethal damage in normal human fibroblasts, Ewing s sarcoma, and human lung adenocarcinoma. No inhibitor (open circles) 8SAB (closed circles). Data points are means 1 standard deviation. Both normal fibroblast and human tumor cell cultures were grown and maintained in Eagle s MEM supplemented with 10% (VA ) fetal bovine serum, streptomycin and penicillin, at S7°C in a humidified chamber of 95% air and 5% CO2. Stock cultures in exponential growth were detached by trypsinization and appropriate cell numbers plated in 25 cm flasks and incubated to permit cell attachment. Inhibitors of poly(ADP-ribose) synthetase (SAB or BZ) were added to the appropriate cultures 2 hr prior to irradiation in air at room temperature. Following irradiation, the cells were exposed to the inhibitors for 24 hr, washed, and replenished with fresh medium. Refeeding was performed every S days and after 10-14 days, cells were fixed and the colonies stained with Giemsa. Only colonies of 50 cells or more were scored as survivors. All experiments were performed in triplicate and the survival data expressed as the mean S.E. of three separate survival experiments.

Culture media and solutions TClOO growth medium TClOO medium supplemented with 10% FCS and 1% penicillin-streptomycin antibiotics (5000 units/ml stock solution). Serum-free TCI 00 Add antibiotics only, as indicated above, hipofectin or similar liposome-mediated transfection reagent. 

Dulbecco s modified Eagle s medium with 10% fetal calf serum (DMEMS) Dulbecco s modified Eagle s medium containing 10% (v/v) fetal calf serum, sodium bicarbonate at 0.3% (w/v) final concentration, L-glutamine at 2 mM final concentration, and penicillin-streptomycin at final concentrations of 100 U and 100 Mg/nil, respectively. To make 500 ml of medium add 50 ml of lOx stock solution of Dulbecco s modified Eagle s medium, 50 ml of fetal calf serum, 20 ml of 7.5% (w/v) sodium bicarbonate, 5 ml of 200 mM L-glutamine, and 5 ml of penicillin-streptomycin (penicillin 1000 U/ml, streptomycin 1000 /ig/ml), and 370 ml of autoclaved water. 

Standard Solution. Weigh an appropriate amount of streptomycin sulfate so that when it is diluted in 0.05 M potassium phosphate buffer, pH 6.0, the resulting stock solution will contain 1000 fig. per milliliter. Keep this stock solution at a temperature of about 15°C. and do not use it for more than 30 days. 

Standard Solution. Prepare the working standard from stock as for streptomycin (Section IV,2). 

For this protocol we employed Fugene 6 as the transfection reagent, so dilutions of DNA stocks and transfection reagent should be done in cell culture medium (DMEM) lacking FBS and penicillin/streptomycin as recommended by supplier. 

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