MATERIALS METHODS 1.1 MATERIALS OF MTT ASSAY The Pin1 transcript and HEK-293 cells were the main components of this experiment. Dulbecco’s Modified Eagle Medium (DMEM) was used for culturing the cells. PEI (Polyethylenimine) was used for both empty (mock) and Pin1 transcript containing vector. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5,diphenyl-tetrazolium bromide(5mg/ml) was used for detection of cell viability. Fetal Bovine Serum (FBS) in 10 %, DMSO and 96 well plate microplate reader were the other materials of the experiment. 1.2 METHODS OF MTT ASSAY The HEK-293 cells were seed into culture medium which was 100 μl. After that, cells were incubated at 37oC and CO2 with 5% during 24 hours. At the end of 24 hours, the plasmid with Pin1 gene was transfected to HEK-293 by the help of PEI reagent and cationic lipid method. Plasmid and PEI reagent were prepared both 1:3 and 1:5 ratios and incubated during 15 minutes at room temperature. Then, these plasmid-PEI mixtures were added to culture and incubate 24 hours and 48 hours for each ratios. The table 1 shows all prepared assay. Then, 10 µl MTT …show more content…
The first one was 1X TBS-T (Tris Buffered Saline) that contains 50 mM Tris (pH 7.5 at RT, pH 8 at +4oC), 150 mM NaCl, 0,1% (v/v) Tween-20. The secon one was the blocking Solution that contains 5% non-fat milk powder dissolved in TBS-T. The third ones were primary and secondary antibody. Also, Antibody dilution is done between 1/1000-1/10000 in 5% non-fat milk dissolved in TBS-T. The last one was the chemiluminescent substrates whose Luminol and peroxide solutions are mixed at same volume to prepare working solution. The Pin1 Antibody (G-8): sc-46660 was used in this experiment which is mouse monoclonal IgG2a. Also, Vinculin (E1E9V) XP® Rabbit mAb #13901 was the monoclonal rabbit antibody with 1:1000 dilution. Moreover, Anti-GFP antibody[9F9.F9](ab1218) was used in this experiment which is Mouse monoclonal