plates wErved the basis of experience. Individual plates were frozen at 24, 48 and 72 hours after the addition, or drugs. DNA content of each well was quantified by F compliance CyQUANT dye manufacturer’s NVP-LDE225 instructions, and absorbance measurement with a fluorimeter CyQUANT. Each condition was performed with six replicates, and the experiment was repeated three times. Detection of phospho Akt and ERK phospho cells were grown to a confluency of 80 serum for 4 h and h with different concentrations of DMSO or NDGA 1 Second, some cultures with 10 nM IGF-I for 15 min removed. The cultures were immediately placed on ice, the medium was removed and the cells were lysed in modified RIPA buffer. Fifty micrograms of protein from each sample were separated by SDS-PAGE and transferred to nitrocellulose. Phosphorylated Akt and total ERK and were detected by immunoblotting. FF dyeing Were stained with propidium iodide apoptotic SH SY5Y cells in DMEM with 10 KK Calf serum culture cultivated.
The cultures were treated with DMSO or NDGA for 24 h. The supernatant was collected in order to hold individual cells. Fixed cells were removed from the plate with trypsin and collected by centrifugation, the supernatant in combination with the cells. The cell pellet was prepared by dropwise addition of cold ethanol carefully produced at 70 fixed vertebrae, and the fourth, the pellet was washed twice and resuspended SP600125 in PBS containing 1 g and emotion Rbt ml propidium iodide. Propidium iodide fluorescence was measured at 30 000 cells per sample using a Becton Dickinson flow- FACSCalibur cytometer. The percentage of cells in each phase of the cell cycle and the percentage of apoptosis of the cells was determined by analysis with ModFit software. The experiment was repeated three times. Detection of caspase-3 cleavage and neuroblastoma cells to confluence 80 h with DMSO or NDGA 6 Alternatively, the cells were grown were cultured serum starved 4th h with or without 60 M NDGA and with or without 10 nM IGF-I for 3 hours Lysates were collected as described above, and the cleavage fragments 17 14 kDa caspase-3 cleaved by SDS-PAGE, immunoblotting with anti-caspase 3 Antique monitoring position established.
Could neuroblastoma Zellmotilit T coated onto gold particles in serum free medium Deckgl fibers at a density of 25,000 cells plated Objekttr engl. Cells were incubated for 2 to erm, incubated the responsibility of the equalization band. The wells were treated with DMSO or 30 M NDGA treated for 1 h. 1 nM IGF-I was then added to some wells. The incubation was continued for 6 hours, followed by fixation in glutaraldehyde 3.5. These are Deckgl Glasobjekttr Mounted loved, then an inverted microscope, which appears t Lietz Orthoplan on a Sony video processor. Digital images of traces of gold in the cells of the three discs separately state ge mag Treated TION protected BEP were collected from 200 using Adobe Photoshop. For each condition were 120 pieces ranging from single cells