Hydrogen Treatment Protects against Cell Death Senescence

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Hydrogen Treatment Protects against Cell Death Senescence ( hydrogen-treatment-protects-against-cell-death-senescence )

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366 Han et al. molecules that overcome its low reaction rates [12]. Hydrogen particles in water that are more than a micrometer in size (i.e., macroparticles) disappear over time. Microparticles of hydrogen that have a diameter <50 μm have important technical applications due to their tendency to decrease in size and collapse in water [13- 15]. Because micro- and macroparticles of hydrogen disappear over time, it is difficult to quantify the exact amount of hydrogen in water; thus, in previous studies, hydrogen water was prepared every week for study. In the present study, we tested various previously published methods to produce hydrogen nanoparticles that are less than 1 μm in diameter and do not evaporate or collapse in water. Because water rich in hydrogen nanoparticles can provide a stable supply of known amounts of hydrogen to cells and tissues, we used it to determine whether hydrogen had an effect on cellular phenotypes associated with aging. Materials and Methods Preparation of Water Enriched in Hydrogen Nanoparticles Various sizes of hydrogen particles were produced with a bubbler made from bamboo (NanoSight; NNB, Korea). Water was pressurized quickly to induce magnetic pressurization, and the particles that were generated first were eliminated to form hydrogen particles of stable sizes. Hydrogen microparticles were then selectively filtered to form hydrogen nanoparticles. In the present study, we prepared water enriched in hydrogen nanoparticles using the method described above in which nearly 70% of total hydrogen nanoparticles had diameters <100 nm. Briefly, the nanobubble generator used consisted of a gas tank and a gas-liquid dispersion system as described in our previous study [16]. The hydrogen gas was fed into the membrane module from the gas tank, by using a gas regulator; the gas pressure was 0.5 MPa. The gas-liquid dispersion system has a membrane module (75 mm long and 20 mm in diameter) with a tubular membrane. The bubble size distribution in nano-scale was measured by the nanoparticle tracking analysis method (NanoSight LM1O- HSBFT14 with 405 nm blue laser; Quantum Design Korea, Korea). Brownian motion of the nanoparticles was analyzed in real-time by a high-sensitivity electron-multiplying CCD camera, based on a laser-illuminated microscopical technique. Cell Isolation and Culture Murine embryonic fibroblasts (MEFs), isolated from embryonic day 12.5 C57BL/6 mouse embryos, were provided from Cefobio (Korea). Cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, USA) containing 10% fetal bovine serum (FBS; GE Healthcare, USA) and 2% penicillin/streptomycin (Sigma- Aldrich, USA). Unless noted otherwise, cells that were passaged twice were used. Quantification of Cell Proliferation Cell proliferation was measured using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Promega, USA) following the manufacturer’s protocol. Briefly, cells were seededin96-wellplatesatadensityof5×10 cellsperwell.Inthe presence or absence of hydroxyurea (10 μmol/ml), cells were incubated with 5 mg/ml MTT for 4 h. The medium was then removed and 150 μl of solubilization solution and stop solution were added to the cells and incubated at 37°C for 4 h. The absorbance of the solution at 570 nm was then measured using a microplate reader. The percentage inhibition of cell growth was calculated as (1 - absorbance of experimental group/absorbance of control group) × 100. Quantification of Cytotoxicity MEFs were seeded in 24-well plates at a density of 5 × 10 cells per well and then incubated in the presence or absence of hydrogen nanoparticle water for 1 to 7 days. Media prepared with water enriched in hydrogen nanoparticles were prepared separately. To quantify cytotoxicity, a lactate dehydrogenase (LDH) Cytotoxicity Assay kit (Cayman Chemical Company, USA) was used, following the manufacturer’s protocol. Briefly, after culturing in 24-well plates to reach passage 2, MEFs were seeded in 96-well plates at a density of 2 × 10 cells per well and grown at 37°C and in 5% CO in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS and 1% penicillin/ streptomycin. After 48 h, 100 μl of the extant medium was transferred to corresponding wells in a new 96-well plate, and 100 μl of reaction solution was added to each well. Plates were incubated at room temperature for 30 min with gentle shaking on an orbital shaker. Absorbance of the solution at 490 nm was measured using a microplate reader. Characterization of Nuclear Morphology Nuclei were observed by fixing MEFs with 4% paraformaldehyde for 20 min at room temperature and post-fixing with 70% ethanol. 4’,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) was diluted in cell culture medium and added at a final concentration of 10 ng/ml for 30 min at room temperature. DAPI-stained nuclei were imaged with a fluorescence microscope (Nikon, Japan). Quantification of β-Galactosidase Staining Senescence-associated β-galactosidase staining was performed using the Senescence β-Galactosidase Staining Kit following the manufacturer’s instructions (Biovision, USA). Briefly, after washing with phosphate-buffered saline (PBS), cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde (Sigma-Aldrich) in PBS for 15 min at room temperature. After washing with PBS, cells were incubated with X-gal staining solution at 37°C for 24 h. Cells were visualized with a light microscope (Nikon) using a 200× objective. The numbers of positive (blue) cells in four random fields of view were counted. J. Microbiol. Biotechnol. 4 3 4 2

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