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Molecules 2011, 16 1397 3.4. TαPcZn-PDT treatment Bel-7402 cells and HDFs in logarithmic growth-phase were seeded in culture plates and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. After being rinsed with phosphate-buffered saline, the cells were treated with TαPcZn stock solution diluted in medium for 2.5 h at 37 °C in the dark. Then the cells were irradiated for 15 min with a SS-B instrument (Wuxi Holyglow Physiotherapy Instrument Co., Ltd., Jiangsu, China) emitting red light within the wavelength range of 600 to 700 nm. The light dose was 53.7 J/cm2 at an irradiance of 59.7 mW/cm2. Thereafter, cells were harvested at 8 h. 3.5. Cell viability assay Cell viability was determined by MTT assay [34]. At 8 h post TαPcZn-PDT of Bel-7402 cells and HDFs, 20 μL of MTT working solution (5 mg/mL MTT in phosphate-buffered saline) was added in 96-well culture plates and incubated continuously at 37 °C for 4 h. All mediums were removed from wells and replaced with 150 μL of DMSO. After the blue-violet crystals were dissolved, the absorbance of each well was measured at 570 nm wavelength with a 680 microplate reader (Bio-Rad, CA, USA). The cell viability was calculated by the formula: cell viability = (absorbance of experimental wells/absorbance of control wells) × 100%. Values are means ±S.D. of three independent experiments. 3.6. Inverted microscope and electron microscopy assay Following a 10.5-h incubation with 54 μM TαPcZn, TαPcZn localization in Bel-7402 cells was assayed by an IX70 inverted microscope (Olympus, Tokyo, Japan). Furthermore, Bel-7402 cells at 8 h post TαPcZn-PDT were also detected by an inverted microscope. After Bel-7402 cells at 8 h post TαPcZn-PDT were fixed with 3% glutaraldehyde in sodium cacodylate buffer (0.1M), they were transferred to phosphate buffer (0.1 M). The cells were then postfixed with 1% osmium tetroxide in S- collidine. After gradient dehydration in ethanol and acetone, the cells were transferred to propylene oxide, and embedded in Epon 812. Semi thin sections were stained with 1% methylene blue. Thereafter, the sample was sectioned into ultrathin slices, contrasted with uranyl acetate and lead citrate, and observed under a JEM1200EX transmission electron microscope (JEOL, Tokyo, Japan) 3.7. Flow cytometry analysis of Annexin V-FITC/PI double stained cells for apoptosis Confirmation of apoptosis was determined by measurement of externalized phosphatidylserine residues as detected using annexin V-FITC. The harvested Bel-7402 cells were collected and washed with ice-cold phosphate-buffered saline, and then suspended in 500 μL of annexin V binding buffer A. 100 μL aliquot was taken, 2 μL of annexin V-FITC and 2 μL of PI were added, and the mixture was incubated for 5 min at room temperature in the dark. After the addition of 400 μL of binding buffer, 1 × 104cells were analyzed on a FACSCAN flow cytometer (Becton Dickinson, San Jose, CA, USA) by using cellquest software. The results are shown as a dotplot graph. In each graph, the percentages of apoptotic cells are indicated in lower right quadrant, the Y–axis corresponds to relative PI staining, and the X-axis corresponds to the log of the FITC signal.PDF Image | Zinc-Mediated Photodynamic Therapy Inhibits the Proliferation
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