ESE staff. However, in most studies, a single Peptidase-4 point in time imaging was performed. We assumed that for the L Ngs imaging at multiple time points w It re useful to consider the dynamic nature of apoptosis induced by drugs. In our previous study, we have genetically UMSCC 22B human head and neck carcinoma modified with a cyclic luciferase, an endogenous marker of apoptosis. Doxil-induced apoptosis was confirmed by imaging several strategies confinement Best of the Lich bioluminescence imaging and diffusion-weighted MRI CONFIRMS. In this study we used L Ngs-PET imaging, the dynamics of cell death by doxorubicin with 18 F as annexin V to monitor contrast media-induced.
Materials and Methods and a cell line of human MTT assay head and neck squamous cell carcinoma cell line UM-SCC 22B was purchased by the University of Michigan and were cultured in medium modified Dulbecco erg Complements Warmth inactivated by 10% f the serum tales bovine serum, 100 units / ml penicillin and 100 g / ml streptomycin at 37 in an atmosphere of 5% CO re second The toxicity t of doxorubicin was Toum 22B SCC cells by a colorimetric test with 3 2, 5 diphenyl tetrazolium bromide determined. All studies were performed with samples in triplicate and repeated at least three times. Briefly, cells were harvested by trypsinization, by in Dulbecco’s modified Eagle, and plated in a 96-well plate at 4,000 or 2,000 cells per well. After treatment Smoothened with various doses of doxorubicin for 48 h the culture medium was replaced and 50 l of 1.0 mg / ml MTT were sterile filtered added to each well. The dye, which has not reacted, was removed after 4 h of incubation, and the formazan crystals were dissolved in 150 l of dimethylsulfoxide St. The absorbance at 570 nm was measured with a multi-mode microplate Leseger t Synergy II. The purification and conjugation of annexin V was annexin V protein expression plasmid kindly provided by Dr. J. Martin Seamus and annexin V-expressed protein was purified and cozy the above-described protocol. The fluorescein isothiocyanate in anhydrous DMSO immediately prior to use gel St, then in annexin V with a molar ratio Ratio of 5:1 was added in 500 l borate buffer.
The mixture is incubated and rotated at room temperature for 60 min for covalent conjugation. The unreacted dye molecules were removed by the PD 10-S Molecules. In Vitro Cell apoptotic F Staining, seeds were in 24-well plates and with doxorubicin for 24 hours. Rin by lacing twice with phosphate-buffered saline Solution, cells with FITC-annexin-V were incubated for 15 min at room temperature. To block 200 g / ml unlabeled annexin V was added 5 min before the FITC-annexin V was added. Then, Hoechst 33342 to the L Solution was added and again for 15 minutes at room temperature. The cells were rinsed three times and observed immediately with a fluorescence microscope. Synthesis of 2 deoxy 2 fluoro radioactive D-glucose Rolipram was purchased by Cardinal Health Nuclear Pharmacy and reconstituted with sterile saline Solution. The average radiochemical purity of the product was 98.5% and the specific activity of t was 91 000 Ci / mmol. N succinimidyl-4 18F fluorobenzoate was synthesized with a modular system as described above. 18F SFB was dissolved in 10 l of acetonitrile, an L Solution and annexin V in 0.1 M NaH again resolved St.