Briefly, DNA was extracted using standard methods and used in a polymerase chain reaction to amplify the entire coding region of the TP53 gene in seven or eight different fragments. The PCR products were screened for mutations using SSCP. Samples showing altered mobility shift in SSCP were further analysed with
direct DNA sequencing to determine the exact RG-7388 location and type of mutation. Cisplatin-induced cell death The cell lines LU-HNSCC 3–8 were harvested by trypsinization, counted and seeded (10,000–26,000 cells/well) in 24-well plates, and allowed to grow for two days as monolayer cultures in DMEM medium (GIBCO, San Diego, CA, USA), supplemented with 10% FBS and antibiotics (100 U/ml streptomycin sulphate, GIBCO), under a 5% CO2 atmosphere at 37°C. On day two, cisplatin (Pharmalink AB, Upplands Väsby, Sweden) was added in serum-free medium, and the cells were incubated for 1 h at concentrations ranging from 0 to 100 μM. Thereafter, the drug-containing medium was removed, and cells were allowed to grow in drug-free medium for 5 days. On day 7, Adavosertib mouse the cell viability was estimated by the crystal violet assay, as described previously . Briefly, the cells were incubated with 0.5% crystal violet (methanol:water,1:4) and excess dye was removed. The cells were solubilized by the addition of 0.10 M citrate
buffer (SIGMA) (50% (v/v) ethanol) and then transferred to a new 96-well plate, and the absorbance was determined spectrophotometrically at 570 nm on a Multiscan MS (Labsystems, Finland) and corrected for background absorbance. 18F-FDG measurements The established cell lines LU-HNSCC 3–8 were harvested
by trypsinization, counted and seeded (50,000–250,000 cells/Petri dish) on day 0. The cells were allowed to grow for two days as monolayer cultures in DMEM medium(GIBCO, San Diego, CA) supplemented with 10% heat-inactivated FBS containing an antibiotic (GIBCO)(100 U/ml streptomycin sulphate), under a 5% CO2 atmosphere new at 37°C. On day three, 2 ml 18F-FDG solution (0.62–1.33 MBq/ml) was added. After an hour the solution was removed by aspiration. The Petri dishes with cells were rinsed three times with PBS. The cells were then harvested from the Petri dishes by trypsinization and neutralized with 4 ml medium, and collected as samples for 18F-FDG determination together with the discarded 18F-FDG solution. The 18F-FDG uptake in the cells and in the washing fractions was estimated using a calibrated 3 x 3 inch NaI(TI) well CP673451 cost counter (in house) (1282 CompuGamma CS, LKB Wallac, Turku, Finland) and all 18F-FDG values were normalized for time. Electronic cell counting was performed using a NucleoCounter™ (Chemotec A/S, Allerod, Denmark) with the NucleoView™ software. The total cell content and number of viable cells were calculated per ml and correlated to the 18F-FDG uptake corrected for decay. This experiment was repeated in a second series.